Proteomes doi: 10.3390/proteomes12010008
Authors: López-Portugués Montes-Bayón Díez
Ovarian cancer is one of the deadliest cancers in women. The lack of specific symptoms, especially at the initial stages of disease development, together with the malignancy heterogeneity, lower the life expectancy of patients. Aiming to improve survival rates, diagnostic and prognostic biomarkers are increasingly employed in clinics, providing gynecologists and oncologists with new tools to guide their treatment decisions. Despite the vast number of investigations, there is still an urgent need to discover more ovarian cancer subtype-specific markers which could further improve patient classification. To this end, high-throughput screening technologies, like mass spectrometry, are applied to deepen the tumoral cellular landscape and describe the malignant phenotypes. As for disease treatment, new targeted therapies, such as those based on PARP inhibitors, have shown great efficacy in destroying the tumoral cells. Likewise, drug-nanocarrier systems targeting the tumoral cells have exhibited promising results. In this narrative review, we summarize the latest achievements in the pursuit of biomarkers for ovarian cancer and recent anti-tumoral therapies.
]]>Proteomes doi: 10.3390/proteomes12010007
Authors: Shahab Mirshahvaladi Nitin Chitranshi Ardeshir Amirkhani Rashi Rajput Devaraj Basavarajappa Roshana Vander Wall Dana Pascovici Angela Godinez Giovanna Galliciotti Joao A. Paulo Veer Gupta Stuart L. Graham Vivek Gupta Mehdi Mirzaei
Neural regeneration and neuroprotection represent strategies for future management of neurodegenerative disorders such as Alzheimer’s disease (AD) or glaucoma. However, the complex molecular mechanisms that are involved in neuroprotection are not clearly understood. A promising candidate that maintains neuroprotective signaling networks is neuroserpin (Serpini1), a serine protease inhibitor expressed in neurons which selectively inhibits extracellular tissue-type plasminogen activator (tPA)/plasmin and plays a neuroprotective role during ischemic brain injury. Abnormal function of this protein has been implicated in several conditions including stroke, glaucoma, AD, and familial encephalopathy with neuroserpin inclusion bodies (FENIB). Here, we explore the potential biochemical roles of Serpini1 by comparing proteome changes between neuroserpin-deficient (NS−/−) and control mice, in the retina (RE), optic nerve (ON), frontal cortex (FC), visual cortex (VC), and cerebellum (CB). To achieve this, a multiple-plex quantitative proteomics approach using isobaric tandem mass tag (TMT) technology was employed followed by functional enrichment and protein–protein interaction analysis. We detected around 5000 proteins in each tissue and a pool of 6432 quantified proteins across all regions, resulting in a pool of 1235 differentially expressed proteins (DEPs). Principal component analysis and hierarchical clustering highlighted similarities and differences in the retina compared to various brain regions, as well as differentiating NS−/− proteome signatures from control samples. The visual cortex revealed the highest number of DEPs, followed by cerebellar regions. Pathway analysis unveiled region-specific changes, including visual perception, focal adhesion, apoptosis, glutamate receptor activation, and supramolecular fiber organization in RE, ON, FC, VC, and CB, respectively. These novel findings provide comprehensive insights into the region-specific networking of Serpini1 in the central nervous system, further characterizing its potential role as a neuroprotective agent. Data are available via ProteomeXchange with identifier PXD046873.
]]>Proteomes doi: 10.3390/proteomes12010006
Authors: Simone König Karin Schork Martin Eisenacher
Many challenges in proteomics result from the high-throughput nature of the experiments. This paper first presents pre-analytical problems, which still occur, although the call for standardization in omics has been ongoing for many years. This article also discusses aspects that affect bioinformatic analysis based on three sets of reference data measured with different orbitrap instruments. Despite continuous advances in mass spectrometer technology as well as analysis software, data-set-wise quality control is still necessary, and decoy-based estimation, although challenged by modern instruments, should be utilized. We draw attention to the fact that numerous young researchers perceive proteomics as a mature, readily applicable technology. However, it is important to emphasize that the maximum potential of the technology can only be realized by an educated handling of its limitations.
]]>Proteomes doi: 10.3390/proteomes12010005
Authors: Hammam H. Said Alan A. Doucette
Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization.
]]>Proteomes doi: 10.3390/proteomes12010004
Authors: Paul Dowling Capucine Trollet Elisa Negroni Dieter Swandulla Kay Ohlendieck
This perspective article is concerned with the question of how proteomics, which is a core technique of systems biology that is deeply embedded in the multi-omics field of modern bioresearch, can help us better understand the molecular pathogenesis of complex diseases. As an illustrative example of a monogenetic disorder that primarily affects the neuromuscular system but is characterized by a plethora of multi-system pathophysiological alterations, the muscle-wasting disease Duchenne muscular dystrophy was examined. Recent achievements in the field of dystrophinopathy research are described with special reference to the proteome-wide complexity of neuromuscular changes and body-wide alterations/adaptations. Based on a description of the current applications of top-down versus bottom-up proteomic approaches and their technical challenges, future systems biological approaches are outlined. The envisaged holistic and integromic bioanalysis would encompass the integration of diverse omics-type studies including inter- and intra-proteomics as the core disciplines for systematic protein evaluations, with sophisticated biomolecular analyses, including physiology, molecular biology, biochemistry and histochemistry. Integrated proteomic findings promise to be instrumental in improving our detailed knowledge of pathogenic mechanisms and multi-system dysfunction, widening the available biomarker signature of dystrophinopathy for improved diagnostic/prognostic procedures, and advancing the identification of novel therapeutic targets to treat Duchenne muscular dystrophy.
]]>Proteomes doi: 10.3390/proteomes12010003
Authors: Diego Fernández-Lázaro Begoña Sanz Jesús Seco-Calvo
Billions of cells die in us every hour, and our tissues do not shrink because there is a natural regulation where Cell Death (CD) is balanced with cell division. The process in which cells eliminate themselves in a controlled manner is called Programmed Cell Death (PCD). The PCD plays an important role during embryonic development, in maintaining homeostasis of the body’s tissues, and in the elimination of damaged cells, under a wide range of physiological and developmental stimuli. A multitude of protein mediators of PCD have been identified and signals have been found to utilize common pathways elucidating the proteins involved. This narrative review focuses on caspase-dependent and caspase-independent PCD pathways. Included are studies of caspase-dependent PCD such as Anoikis, Catastrophe Mitotic, Pyroptosis, Emperitosis, Parthanatos and Cornification, and Caspase-Independent PCD as Wallerian Degeneration, Ferroptosis, Paraptosis, Entosis, Methuosis, and Extracellular Trap Abnormal Condition (ETosis), as well as neutrophil extracellular trap abnormal condition (NETosis) and Eosinophil Extracellular Trap Abnormal Condition (EETosis). Understanding PCD from those reported in this review could shed substantial light on the processes of biological homeostasis. In addition, identifying specific proteins involved in these processes is mandatory to identify molecular biomarkers, as well as therapeutic targets. This knowledge could provide the ability to modulate the PCD response and could lead to new therapeutic interventions in a wide range of diseases.
]]>Proteomes doi: 10.3390/proteomes12010002
Authors: Andreas Burkovski
Within the genus Corynebacterium, six species are potential carriers of the tox gene, which encodes the highly potent diphtheria exotoxin: Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis and Corynebacterium silvaticum. Based on their potential to infect different host species and cause either human infections, zoonotic diseases or infections of economically important animals, these bacteria are of high scientific and economic interest and different research groups have carried out proteome analyses. These showed that especially the combination of MS-based proteomics with bioinformatic tools helped significantly to elucidate the functional aspects of corynebacterial genomes and to handle the genome and proteome complexity. The combination of proteomic and bioinformatic approaches was also used to discover new vaccine and drug targets. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been established as a fast and precise tool for the identification of these bacteria.
]]>Proteomes doi: 10.3390/proteomes12010001
Authors: Yan Feng Qingji Huo Bai-Yan Li Hiroki Yokota
This review covers the diagnostic potential of urinary biomarkers, shedding light on their linkage to cancer progression. Urinary biomarkers offer non-invasive avenues for detecting cancers, potentially bypassing the invasiveness of biopsies. The investigation focuses primarily on breast and prostate cancers due to their prevalence among women and men, respectively. The intricate interplay of urinary proteins is explored, revealing a landscape where proteins exhibit context-dependent behaviors. The review highlights the potential impact of physical activity on urinary proteins, suggesting its influence on tumorigenic behaviors. Exercise-conditioned urine may emerge as a potential diagnostic biomarker source. Furthermore, treatment effects, notably after lumpectomy and prostatectomy, induce shifts in the urinary proteome, indicating therapeutic impacts rather than activating oncogenic signaling. The review suggests further investigations into the double-sided, context-dependent nature of urinary proteins, the potential role of post-translational modifications (PTM), and the integration of non-protein markers like mRNA and metabolites. It also discusses a linkage of urinary proteomes with secretomes from induced tumor-suppressing cells (iTSCs). Despite challenges like cancer heterogeneity and sample variability due to age, diet, and comorbidities, harnessing urinary proteins and proteoforms may hold promise for advancing our understanding of cancer progressions, as well as the diagnostic and therapeutic role of urinary proteins.
]]>Proteomes doi: 10.3390/proteomes11040039
Authors: Mariia Zmyslia Klemens Fröhlich Trinh Dao Alexander Schmidt Claudia Jessen-Trefzer
Understanding the complex mechanisms of mycobacterial pathophysiology and adaptive responses presents challenges that can hinder drug development. However, employing physiologically relevant conditions, such as those found in human macrophages or simulating physiological growth conditions, holds promise for more effective drug screening. A valuable tool in this pursuit is proteomics, which allows for a comprehensive analysis of adaptive responses. In our study, we focused on Mycobacterium smegmatis, a model organism closely related to the pathogenic Mycobacterium tuberculosis, to investigate the impact of various carbon sources on mycobacterial growth. To facilitate this research, we developed a cost-effective, straightforward, and high-quality pipeline for proteome analysis and compared six different carbon source conditions. Additionally, we have created an online tool to present and analyze our data, making it easily accessible to the community. This user-friendly platform allows researchers and interested parties to explore and interpret the results effectively. Our findings shed light on mycobacterial adaptive physiology and present potential targets for drug development, contributing to the fight against tuberculosis.
]]>Proteomes doi: 10.3390/proteomes11040038
Authors: Madhvi Sharma Amanpreet K. Sidhu Mahesh Kumar Samota Mamta Gupta Pushpendra Koli Mukesh Choudhary
Abiotic stresses profoundly alter plant growth and development, resulting in yield losses. Plants have evolved adaptive mechanisms to combat these challenges, triggering intricate molecular responses to maintain tissue hydration and temperature stability during stress. A pivotal player in this defense is histone modification, governing gene expression in response to diverse environmental cues. Post-translational modifications (PTMs) of histone tails, including acetylation, phosphorylation, methylation, ubiquitination, and sumoylation, regulate transcription, DNA processes, and stress-related traits. This review comprehensively explores the world of PTMs of histones in plants and their vital role in imparting various abiotic stress tolerance in plants. Techniques, like chromatin immune precipitation (ChIP), ChIP-qPCR, mass spectrometry, and Cleavage Under Targets and Tag mentation, have unveiled the dynamic histone modification landscape within plant cells. The significance of PTMs in enhancing the plants’ ability to cope with abiotic stresses has also been discussed. Recent advances in PTM research shed light on the molecular basis of stress tolerance in plants. Understanding the intricate proteome complexity due to various proteoforms/protein variants is a challenging task, but emerging single-cell resolution techniques may help to address such challenges. The review provides the future prospects aimed at harnessing the full potential of PTMs for improved plant responses under changing climate change.
]]>Proteomes doi: 10.3390/proteomes11040037
Authors: Yulia Merkher Elizaveta Kontareva Anastasia Alexandrova Rajesha Javaraiah Margarita Pustovalova Sergey Leonov
Flaxseed has been recognized as a valuable source of nutrients and bioactive compounds, including proteins that possess various health benefits. In recent years, studies have shown that flaxseed proteins, including albumins, globulins, glutelin, and prolamins, possess anti-cancer properties. These properties are attributed to their ability to inhibit cancer cell proliferation, induce apoptosis, and interfere with cancer cell signaling pathways, ultimately leading to the inhibition of metastasis. Moreover, flaxseed proteins have been reported to modulate cancer cell mechanobiology, leading to changes in cell behavior and reduced cancer cell migration and invasion. This review provides an overview of the anti-cancer properties of flaxseed proteins, with a focus on their potential use in cancer treatment. Additionally, it highlights the need for further research to fully establish the potential of flaxseed proteins in cancer therapy.
]]>Proteomes doi: 10.3390/proteomes11040036
Authors: Morteza Abyadeh Vivek Gupta Xinyue Liu Valentina Rossio Mehdi Mirzaei Jennifer Cornish Joao A. Paulo Paul A. Haynes
Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein–protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.
]]>Proteomes doi: 10.3390/proteomes11040035
Authors: Ana Montero-Calle María Garranzo-Asensio Raquel Rejas-González Jaime Feliu Marta Mendiola Alberto Peláez-García Rodrigo Barderas
The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = −45 and CV = −60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.
]]>Proteomes doi: 10.3390/proteomes11040034
Authors: Tonci Ivanisevic Raj N. Sewduth
Multi-omics is a cutting-edge approach that combines data from different biomolecular levels, such as DNA, RNA, proteins, metabolites, and epigenetic marks, to obtain a holistic view of how living systems work and interact. Multi-omics has been used for various purposes in biomedical research, such as identifying new diseases, discovering new drugs, personalizing treatments, and optimizing therapies. This review summarizes the latest progress and challenges of multi-omics for designing new treatments for human diseases, focusing on how to integrate and analyze multiple proteome data and examples of how to use multi-proteomics data to identify new drug targets. We also discussed the future directions and opportunities of multi-omics for developing innovative and effective therapies by deciphering proteome complexity.
]]>Proteomes doi: 10.3390/proteomes11040033
Authors: Mukul Jain Rupal Dhariwal Nil Patil Sandhya Ojha Reshma Tendulkar Mugdha Tendulkar Parmdeep Singh Dhanda Alpa Yadav Prashant Kaushik
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by progressive cognitive decline and memory loss. Early and accurate diagnosis of AD is crucial for implementing timely interventions and developing effective therapeutic strategies. Proteome-based biomarkers have emerged as promising tools for AD diagnosis and prognosis due to their ability to reflect disease-specific molecular alterations. There is of great significance for biomarkers in AD diagnosis and management. It emphasizes the limitations of existing diagnostic approaches and the need for reliable and accessible biomarkers. Proteomics, a field that comprehensively analyzes the entire protein complement of cells, tissues, or bio fluids, is presented as a powerful tool for identifying AD biomarkers. There is a diverse range of proteomic approaches employed in AD research, including mass spectrometry, two-dimensional gel electrophoresis, and protein microarrays. The challenges associated with identifying reliable biomarkers, such as sample heterogeneity and the dynamic nature of the disease. There are well-known proteins implicated in AD pathogenesis, such as amyloid-beta peptides, tau protein, Apo lipoprotein E, and clusterin, as well as inflammatory markers and complement proteins. Validation and clinical utility of proteome-based biomarkers are addressing the challenges involved in validation studies and the diagnostic accuracy of these biomarkers. There is great potential in monitoring disease progression and response to treatment, thereby aiding in personalized medicine approaches for AD patients. There is a great role for bioinformatics and data analysis in proteomics for AD biomarker research and the importance of data preprocessing, statistical analysis, pathway analysis, and integration of multi-omics data for a comprehensive understanding of AD pathophysiology. In conclusion, proteome-based biomarkers hold great promise in the field of AD research. They provide valuable insights into disease mechanisms, aid in early diagnosis, and facilitate personalized treatment strategies. However, further research and validation studies are necessary to harness the full potential of proteome-based biomarkers in clinical practice.
]]>Proteomes doi: 10.3390/proteomes11040032
Authors: Liang Jin Fei Wang Xue Wang Bohdan P. Harvey Yingtao Bi Chenqi Hu Baoliang Cui Anhdao T. Darcy John W. Maull Ben R. Phillips Youngjae Kim Gary J. Jenkins Thierry R. Sornasse Yu Tian
Rheumatoid arthritis (RA) is a systemic autoimmune and inflammatory disease. Plasma biomarkers are critical for understanding disease mechanisms, treatment effects, and diagnosis. Mass spectrometry-based proteomics is a powerful tool for unbiased biomarker discovery. However, plasma proteomics is significantly hampered by signal interference from high-abundance proteins, low overall protein coverage, and high levels of missing data from data-dependent acquisition (DDA). To achieve quantitative proteomics analysis for plasma samples with a balance of throughput, performance, and cost, we developed a workflow incorporating plate-based high abundance protein depletion and sample preparation, comprehensive peptide spectral library building, and data-independent acquisition (DIA) SWATH mass spectrometry-based methodology. In this study, we analyzed plasma samples from both RA patients and healthy donors. The results showed that the new workflow performance exceeded that of the current state-of-the-art depletion-based plasma proteomic platforms in terms of both data quality and proteome coverage. Proteins from biological processes related to the activation of systemic inflammation, suppression of platelet function, and loss of muscle mass were enriched and differentially expressed in RA. Some plasma proteins, particularly acute-phase reactant proteins, showed great power to distinguish between RA patients and healthy donors. Moreover, protein isoforms in the plasma were also analyzed, providing even deeper proteome coverage. This workflow can serve as a basis for further application in discovering plasma biomarkers of other diseases.
]]>Proteomes doi: 10.3390/proteomes11040031
Authors: Yueh-Chung Chen Chen-Chung Liao Hao-Ai Shui Pei-Hsuan Huang Li-Jane Shih
Trophoblast migration and invasion play crucial roles in placental development. However, the effects of (-)-epigallocatechin-3-gallate (EGCG) on trophoblast cell functions remain largely unexplored. In this study, we investigated the impact of EGCG on the survival of trophoblast cells and employed a proteomics analysis to evaluate its influence on trophoblast cell migration and invasion. Be-Wo trophoblast cells were treated with EGCG, and a zone closure assay was conducted to assess the cell migration and invasion. Subsequently, a proteomics analysis was performed on the treated and control groups, followed by a bioinformatics analysis to evaluate the affected biological pathways and protein networks. A quantitative real-time PCR and Western blot analysis were carried out to validate the proteomics findings. Our results showed that EGCG significantly suppressed the trophoblast migration and invasion at a concentration not affecting cell survival. The proteomics analysis revealed notable differences in the protein expression between the EGCG-treated and control groups. Specifically, EGCG downregulated the signaling pathways related to EIF2, mTOR, and estrogen response, as well as the processes associated with the cytoskeleton, extracellular matrix, and protein translation. Conversely, EGCG upregulated the pathways linked to lipid degradation and oxidative metabolism. The quantitative PCR showed that EGCG modulated protein expression by regulating gene transcription, and the Western blot analysis confirmed its impact on cytoskeleton and extracellular matrix reorganization. These findings suggest EGCG may inhibit trophoblast migration and invasion through multiple signaling pathways, highlighting the potential risks associated with consuming EGCG-containing products during pregnancy. Future research should investigate the impact of EGCG intake on maternal and fetal proteoforms.
]]>Proteomes doi: 10.3390/proteomes11040030
Authors: Valentina Rossio Xinyue Liu Joao A. Paulo
The yeast Saccharomyces cerevisiae is a powerful model system that is often used to expand our understanding of cellular processes and biological functions. Although many genetically well-characterized laboratory strains of S. cerevisiae are available, they may have different genetic backgrounds which can confound data interpretation. Here, we report a comparative whole-proteome analysis of two common laboratory yeast background strains, W303 and BY4742, in both exponential and stationary growth phases using isobaric-tag-based mass spectrometry to highlight differences in proteome complexity. We quantified over 4400 proteins, hundreds of which showed differences in abundance between strains and/or growth phases. Moreover, we used proteome-wide protein abundance to profile the mating type of the strains used in the experiment, the auxotrophic markers, and associated metabolic pathways, as well as to investigate differences in particular classes of proteins, such as the pleiotropic drug resistance (PDR) proteins. This study is a valuable resource that offers insight into mechanistic differences between two common yeast background strains and can be used as a guide to select a background that is best suited for addressing a particular biological question.
]]>Proteomes doi: 10.3390/proteomes11040029
Authors: Ireshyn Selvan Govender Rethabile Mokoena Stoyan Stoychev Previn Naicker
Urine provides a diverse source of information related to a patient’s health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.
]]>Proteomes doi: 10.3390/proteomes11040028
Authors: Valentina Rossio Joao A. Paulo
The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that, while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that, under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, implying harvesting-method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked, aspect of experimental design.
]]>Proteomes doi: 10.3390/proteomes11030027
Authors: Jude Jessie Bond Gordon Refshauge Matthew T. Newell Benjamin W. B. Holman David Wheeler Serey Woodgate Karthik S. Kamath Richard C. Hayes
The value of crops such as perennial wheat (PW) for grain and grazing compared to conventional wheat (W), or the addition of lucerne to PW (PWL) is still being determined. This research sought to determine if these diets were associated with changes in the membranebound proteins that transport nutrients in the rumen epithelium (RE). Crossbred ewes (Poll Dorset × Merino) were fed W, PW, or PWL (50:50) fresh-cut forage ad libitum for 4 weeks. Average daily gain (ADG; p < 0.001) was highest in the W-fed lambs compared to the PW and PWL. Metabolisable energy intake (MEI) was higher in lambs fed W (p < 0.001) compared to PW and PWL. In pairwise comparisons of the PW and PWL diet group we found protein abundance was significantly (p < 0.05, FDR < 0.05, Benjamini p < 0.05) different in fatty acid metabolism, oxidative phosphorylation, and biosynthesis of cofactors pathways. There were not any differences in protein abundance related to nutrient transport or energy metabolism in the RE between W- vs. PW- and W- vs. PWL-fed lambs. However, in the PW- vs. PWL-fed lambs, there was a difference in the level of proteins regulating the metabolism of fatty acids and energy production in the mitochondria of the rumen epithelium.
]]>Proteomes doi: 10.3390/proteomes11030026
Authors: Marianna Zolotovskaia Maks Kovalenko Polina Pugacheva Victor Tkachev Alexander Simonov Maxim Sorokin Alexander Seryakov Andrew Garazha Nurshat Gaifullin Marina Sekacheva Galina Zakharova Anton A. Buzdin
Individual gene expression and molecular pathway activation profiles were shown to be effective biomarkers in many cancers. Here, we used the human interactome model to algorithmically build 7470 molecular pathways centered around individual gene products. We assessed their associations with tumor type and survival in comparison with the previous generation of molecular pathway biomarkers (3022 “classical” pathways) and with the RNA transcripts or proteomic profiles of individual genes, for 8141 and 1117 samples, respectively. For all analytes in RNA and proteomic data, respectively, we found a total of 7441 and 7343 potential biomarker associations for gene-centric pathways, 3020 and 2950 for classical pathways, and 24,349 and 6742 for individual genes. Overall, the percentage of RNA biomarkers was statistically significantly higher for both types of pathways than for individual genes (p < 0.05). In turn, both types of pathways showed comparable performance. The percentage of cancer-type-specific biomarkers was comparable between proteomic and transcriptomic levels, but the proportion of survival biomarkers was dramatically lower for proteomic data. Thus, we conclude that pathway activation level is the advanced type of biomarker for RNA and proteomic data, and momentary algorithmic computer building of pathways is a new credible alternative to time-consuming hypothesis-driven manual pathway curation and reconstruction.
]]>Proteomes doi: 10.3390/proteomes11030025
Authors: Sam Hobson Emmanouil Mavrogeorgis Tianlin He Justyna Siwy Thomas Ebert Karolina Kublickiene Peter Stenvinkel Harald Mischak
Given the pathophysiological continuum of chronic kidney disease (CKD), different molecular determinants affecting progression may be associated with distinct disease phases; thus, identification of these players are crucial for guiding therapeutic decisions, ideally in a non-invasive, repeatable setting. Analyzing the urinary peptidome has been proven an efficient method for biomarker determination in CKD, among other diseases. In this work, after applying several selection criteria, urine samples from 317 early (stage 2) and advanced (stage 3b–5) CKD patients were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS). The entire two groups were initially compared to highlight the respective pathophysiology between initial and late disease phases. Subsequently, slow and fast progressors were compared within each group in an attempt to distinguish phase-specific disease progression molecules. The early vs. late-stage CKD comparison revealed 929 significantly different peptides, most of which were downregulated and 268 with collagen origins. When comparing slow vs. fast progressors in early stage CKD, 42 peptides were significantly altered, 30 of which were collagen peptide fragments. This association suggests the development of structural changes may be reversible at an early stage. The study confirms previous findings, based on its multivariable-matched progression groups derived from a large initial cohort. However, only four peptide fragments differed between slow vs. fast progressors in late-stage CKD, indicating different pathogenic processes occur in fast and slow progressors in different stages of CKD. The defined peptides associated with CKD progression at early stage might potentially constitute a non-invasive approach to improve patient management by guiding (personalized) intervention.
]]>Proteomes doi: 10.3390/proteomes11030024
Authors: Luís Ramalhete Emanuel Vigia Rúben Araújo Hugo Pinto Marques
Pancreatic cancer is a devastating disease that has a grim prognosis, highlighting the need for improved screening, diagnosis, and treatment strategies. Currently, the sole biomarker for pancreatic ductal adenocarcinoma (PDAC) authorized by the U.S. Food and Drug Administration is CA 19-9, which proves to be the most beneficial in tracking treatment response rather than in early detection. In recent years, proteomics has emerged as a powerful tool for advancing our understanding of pancreatic cancer biology and identifying potential biomarkers and therapeutic targets. This review aims to offer a comprehensive survey of proteomics’ current status in pancreatic cancer research, specifically accentuating its applications and its potential to drastically enhance screening, diagnosis, and treatment response. With respect to screening and diagnostic precision, proteomics carries the capacity to augment the sensitivity and specificity of extant screening and diagnostic methodologies. Nonetheless, more research is imperative for validating potential biomarkers and establishing standard procedures for sample preparation and data analysis. Furthermore, proteomics presents opportunities for unveiling new biomarkers and therapeutic targets, as well as fostering the development of personalized treatment strategies based on protein expression patterns associated with treatment response. In conclusion, proteomics holds great promise for advancing our understanding of pancreatic cancer biology and improving patient outcomes. It is essential to maintain momentum in investment and innovation in this arena to unearth more groundbreaking discoveries and transmute them into practical diagnostic and therapeutic strategies in the clinical context.
]]>Proteomes doi: 10.3390/proteomes11030023
Authors: Dianny Elizabeth Jimenez Muhammad Tahir Muhammad Faheem Wellington Bruno dos Santos Alves Barbara de Lucena Correa Gabriel Rocha de Andrade Martin R. Larsen Getulio Pereira de Oliveira Rinaldo Wellerson Pereira
In recent decades, the role played by extracellular vesicles in physiological and pathological processes has attracted attention. Extracellular vesicles are released by different types of cells and carry molecules that could become biomarkers for the diagnosis of diseases. Extracellular vesicles are also moldable tools for the controlled release of bioactive substances in clinical and therapeutic applications. However, one of the significant challenges when studying these exciting and versatile vesicles is the purification process, which presents significant difficulties in terms of lack of purity, yield, and reproducibility, reflected in unreliable data. Therefore, our objective in the present study was to compare the proteomic profile of serum-derived EVs purified using ExoQuick™ (Systems Biosciences), Total Isolation Kit (Life Technologies), Ultracentrifugation, and Ultrafiltration. Each technique utilized for purification has shown different concentrations and populations of purified particles. The results showed marked differences in distribution, size, and protein content, demonstrating the need to develop reproducible and reliable protocols to isolate extracellular vesicles for their clinical application.
]]>Proteomes doi: 10.3390/proteomes11030022
Authors: Eduardo Alvarez-Rivera Emanuel J. Ortiz-Hernández Elyette Lugo Lorraine M. Lozada-Reyes Nawal M. Boukli
Recent advances in the field of proteomics have allowed extensive insights into the molecular regulations of the cell proteome. Specifically, this allows researchers to dissect a multitude of signaling arrays while targeting for the discovery of novel protein signatures. These approaches based on data mining are becoming increasingly powerful for identifying both potential disease mechanisms as well as indicators for disease progression and overall survival predictive and prognostic molecular markers for cancer. Furthermore, mass spectrometry (MS) integrations satisfy the ongoing demand for in-depth biomarker validation. For the purpose of this review, we will highlight the current developments based on MS sensitivity, to place quantitative proteomics into clinical settings and provide a perspective to integrate proteomics data for future applications in cancer precision medicine. We will also discuss malignancies associated with oncogenic viruses such as Acquire Immunodeficiency Syndrome (AIDS) and suggest novel mechanisms behind this phenomenon. Human Immunodeficiency Virus type-1 (HIV-1) proteins are known to be oncogenic per se, to induce oxidative and endoplasmic reticulum stresses, and to be released from the infected or expressing cells. HIV-1 proteins can act alone or in collaboration with other known oncoproteins, which cause the bulk of malignancies in people living with HIV-1 on ART.
]]>Proteomes doi: 10.3390/proteomes11020021
Authors: Despoina P. Kiouri Charalampos Ntallis Konstantinos Kelaidonis Massimiliano Peana Sotirios Tsiodras Thomas Mavromoustakos Alessandro Giuliani Harry Ridgway Graham J. Moore John M. Matsoukas Christos T. Chasapis
The potential of targeting the Renin-Angiotensin-Aldosterone System (RAAS) as a treatment for the coronavirus disease 2019 (COVID-19) is currently under investigation. One way to combat this disease involves the repurposing of angiotensin receptor blockers (ARBs), which are antihypertensive drugs, because they bind to angiotensin-converting enzyme 2 (ACE2), which in turn interacts with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. However, there has been no in silico analysis of the potential toxicity risks associated with the use of these drugs for the treatment of COVID-19. To address this, a network-based bioinformatics methodology was used to investigate the potential side effects of known Food and Drug Administration (FDA)-approved antihypertensive drugs, Sartans. This involved identifying the human proteins targeted by these drugs, their first neighbors, and any drugs that bind to them using publicly available experimentally supported data, and subsequently constructing proteomes and protein–drug interactomes. This methodology was also applied to Pfizer’s Paxlovid, an antiviral drug approved by the FDA for emergency use in mild-to-moderate COVID-19 treatment. The study compares the results for both drug categories and examines the potential for off-target effects, undesirable involvement in various biological processes and diseases, possible drug interactions, and the potential reduction in drug efficiency resulting from proteoform identification.
]]>Proteomes doi: 10.3390/proteomes11020020
Authors: Jason Linzer Zachary Phelps Shivasuryan Vummidi Bo Young Elizabeth Lee Nicolas Coant John D. Haley
Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.
]]>Proteomes doi: 10.3390/proteomes11020019
Authors: Isabel De Figueiredo Bernard Bartenlian Guillaume Van der Rest Antoine Pallandre Frédéric Halgand
Protein biomarkers have been the subject of intensive studies as a target for disease diagnostics and monitoring. Indeed, biomarkers have been extensively used for personalized medicine. In biological samples, these biomarkers are most often present in low concentrations masked by a biologically complex proteome (e.g., blood) making their detection difficult. This complexity is further increased by the needs to detect proteoforms and proteome complexity such as the dynamic range of compound concentrations. The development of techniques that simultaneously pre-concentrate and identify low-abundance biomarkers in these proteomes constitutes an avant-garde approach to the early detection of pathologies. Chromatographic-based methods are widely used for protein separation, but these methods are not adapted for biomarker discovery, as they require complex sample handling due to the low biomarker concentration. Therefore, microfluidics devices have emerged as a technology to overcome these shortcomings. In terms of detection, mass spectrometry (MS) is the standard analytical tool given its high sensitivity and specificity. However, for MS, the biomarker must be introduced as pure as possible in order to avoid chemical noise and improve sensitivity. As a result, microfluidics coupled with MS has become increasingly popular in the field of biomarker discovery. This review will show the different approaches to protein enrichment using miniaturized devices and the importance of their coupling with MS.
]]>Proteomes doi: 10.3390/proteomes11020018
Authors: Shipan Fan Ansgar Poetsch
Extracellular vesicles (EVs), the lipid bilayer membranous structures of particles, are produced and released from almost all cells, including eukaryotes and prokaryotes. The versatility of EVs has been investigated in various pathologies, including development, coagulation, inflammation, immune response modulation, and cell–cell communication. Proteomics technologies have revolutionized EV studies by enabling high-throughput analysis of their biomolecules to deliver comprehensive identification and quantification with rich structural information (PTMs, proteoforms). Extensive research has highlighted variations in EV cargo depending on vesicle size, origin, disease, and other features. This fact has sparked activities to use EVs for diagnosis and treatment to ultimately achieve clinical translation with recent endeavors summarized and critically reviewed in this publication. Notably, successful application and translation require a constant improvement of methods for sample preparation and analysis and their standardization, both of which are areas of active research. This review summarizes the characteristics, isolation, and identification approaches for EVs and the recent advances in EVs for clinical biofluid analysis to gain novel knowledge by employing proteomics. In addition, the current and predicted future challenges and technical barriers are also reviewed and discussed.
]]>Proteomes doi: 10.3390/proteomes11020017
Authors: Klára Brožová Brigitte Hantusch Lukas Kenner Klaus Kratochwill
Breast cancer (BC) is a major global health issue, affecting a significant proportion of the female population and contributing to high rates of mortality. One of the primary challenges in the treatment of BC is the disease’s heterogeneity, which can lead to ineffective therapies and poor patient outcomes. Spatial proteomics, which involves the study of protein localization within cells, offers a promising approach for understanding the biological processes that contribute to cellular heterogeneity within BC tissue. To fully leverage the potential of spatial proteomics, it is critical to identify early diagnostic biomarkers and therapeutic targets, and to understand protein expression levels and modifications. The subcellular localization of proteins is a key factor in their physiological function, making the study of subcellular localization a major challenge in cell biology. Achieving high resolution at the cellular and subcellular level is essential for obtaining an accurate spatial distribution of proteins, which in turn can enable the application of proteomics in clinical research. In this review, we present a comparison of current methods of spatial proteomics in BC, including untargeted and targeted strategies. Untargeted strategies enable the detection and analysis of proteins and peptides without a predetermined molecular focus, whereas targeted strategies allow the investigation of a predefined set of proteins or peptides of interest, overcoming the limitations associated with the stochastic nature of untargeted proteomics. By directly comparing these methods, we aim to provide insights into their strengths and limitations and their potential applications in BC research.
]]>Proteomes doi: 10.3390/proteomes11020016
Authors: Neha Varshney Abhinava K. Mishra
Protein phosphorylation is a key post-translational modification (PTM) that is a central regulatory mechanism of many cellular signaling pathways. Several protein kinases and phosphatases precisely control this biochemical process. Defects in the functions of these proteins have been implicated in many diseases, including cancer. Mass spectrometry (MS)-based analysis of biological samples provides in-depth coverage of phosphoproteome. A large amount of MS data available in public repositories has unveiled big data in the field of phosphoproteomics. To address the challenges associated with handling large data and expanding confidence in phosphorylation site prediction, the development of many computational algorithms and machine learning-based approaches have gained momentum in recent years. Together, the emergence of experimental methods with high resolution and sensitivity and data mining algorithms has provided robust analytical platforms for quantitative proteomics. In this review, we compile a comprehensive collection of bioinformatic resources used for the prediction of phosphorylation sites, and their potential therapeutic applications in the context of cancer.
]]>Proteomes doi: 10.3390/proteomes11020015
Authors: Abraham Josué Nevárez-Ramírez Ana Laura Guzmán-Ortiz Pedro Cortes-Reynosa Eduardo Perez-Salazar Gustavo Alberto Jaimes-Ortega Ricardo Valle-Rios Álvaro Marín-Hernández José S. Rodríguez-Zavala Eliel Ruiz-May José Luis Castrejón-Flores Héctor Quezada
Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.
]]>Proteomes doi: 10.3390/proteomes11020014
Authors: Miguel Rodrigues Maria José López-Martinez Alba Ortin-Bustillo Jose Joaquin Cerón Silvia Martinez-Subiela Alberto Muñoz-Prieto Elsa Lamy
Escherichia coli represents the main cause of diarrhoea in pigs. Saliva can provide information about the pathophysiology of diseases and be a source of biomarkers. We aimed to identify changes in the salivary proteome of pigs with diarrhoea caused by E. coli. Saliva samples were collected from 10 pigs with this disease and 10 matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. For validation, adenosine deaminase (ADA) was measured in 28 healthy and 28 diseased pigs. In 1DE, increases in lipocalin and IgA bands were observed for diseased pigs, whereas bands containing proteins such as odorant-binding protein and/or prolactin-inducible protein presented decreased concentrations. Two-dimensional gel electrophoresis (2DE) results showed that saliva from E. coli animals presented higher expression levels of lipocalin, ADA, IgA and albumin peptides, being ADA activity increased in the diseased pigs in the validation study. Spots containing alpha-amylase, carbonic anhydrase VI, and whole albumin were decreased in diseased animals. Overall, pigs with diarrhoea caused by E. coli have changes in proteins in their saliva related to various pathophysiological mechanisms such as inflammation and immune function and could potentially be biomarkers of this disease.
]]>Proteomes doi: 10.3390/proteomes11020013
Authors: Anca-Narcisa Neagu Danielle Whitham Logan Seymour Norman Haaker Isabella Pelkey Costel C. Darie
Invasive ductal carcinoma (IDC) is the most common histological subtype of malignant breast cancer (BC), and accounts for 70–80% of all invasive BCs. IDC demonstrates great heterogeneity in clinical and histopathological characteristics, prognoses, treatment strategies, gene expressions, and proteomic profiles. Significant proteomic determinants of the progression from intraductal pre-invasive malignant lesions of the breast, which characterize a ductal carcinoma in situ (DCIS), to IDC, are still poorly identified, validated, and clinically applied. In the era of “6P” medicine, it remains a great challenge to determine which patients should be over-treated versus which need to be actively monitored without aggressive treatment. The major difficulties for designating DCIS to IDC progression may be solved by understanding the integrated genomic, transcriptomic, and proteomic bases of invasion. In this review, we showed that multiple proteomics-based techniques, such as LC–MS/MS, MALDI-ToF MS, SELDI-ToF-MS, MALDI-ToF/ToF MS, MALDI-MSI or MasSpec Pen, applied to in-tissue, off-tissue, BC cell lines and liquid biopsies, improve the diagnosis of IDC, as well as its prognosis and treatment monitoring. Classic proteomics strategies that allow the identification of dysregulated protein expressions, biological processes, and interrelated pathway analyses based on aberrant protein–protein interaction (PPI) networks have been improved to perform non-invasive/minimally invasive biomarker detection of early-stage IDC. Thus, in modern surgical oncology, highly sensitive, rapid, and accurate MS-based detection has been coupled with “proteome point sampling” methods that allow for proteomic profiling by in vivo “proteome point characterization”, or by minimal tissue removal, for ex vivo accurate differentiation and delimitation of IDC. For the detection of low-molecular-weight proteins and protein fragments in bodily fluids, LC–MS/MS and MALDI-MS techniques may be coupled to enrich and capture methods which allow for the identification of early-stage IDC protein biomarkers that were previously invisible for MS-based techniques. Moreover, the detection and characterization of protein isoforms, including posttranslational modifications of proteins (PTMs), is also essential to emphasize specific molecular mechanisms, and to assure the early-stage detection of IDC of the breast.
]]>Proteomes doi: 10.3390/proteomes11020012
Authors: Liting Deng Vivek Gupta Morteza Abyadeh Nitin Chitranshi Kanishka Pushpitha Yunqi Wu Veer Gupta Yuyi You Joao A. Paulo Stuart L. Graham Mehdi Mirzaei Paul A. Haynes
Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H2O2 at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.
]]>Proteomes doi: 10.3390/proteomes11020011
Authors: Carmen Bedia Nuria Dalmau Lars K. Nielsen Romà Tauler Igor Marín de Mas
Although numerous studies support a dose–effect relationship between Endocrine disruptors (EDs) and the progression and malignancy of tumors, the impact of a chronic exposure to non-lethal concentrations of EDs in cancer remains unknown. More specifically, a number of studies have reported the impact of Aldrin on a variety of cancer types, including prostate cancer. In previous studies, we demonstrated the induction of the malignant phenotype in DU145 prostate cancer (PCa) cells after a chronic exposure to Aldrin (an ED). Proteins are pivotal in the regulation and control of a variety of cellular processes. However, the mechanisms responsible for the impact of ED on PCa and the role of proteins in this process are not yet well understood. Here, two complementary computational approaches have been employed to investigate the molecular processes underlying the acquisition of malignancy in prostate cancer. First, the metabolic reprogramming associated with the chronic exposure to Aldrin in DU145 cells was studied by integrating transcriptomics and metabolomics via constraint-based metabolic modeling. Second, gene set enrichment analysis was applied to determine (i) altered regulatory pathways and (ii) the correlation between changes in the transcriptomic profile of Aldrin-exposed cells and tumor progression in various types of cancer. Experimental validation confirmed predictions revealing a disruption in metabolic and regulatory pathways. This alteration results in the modification of protein levels crucial in regulating triacylglyceride/cholesterol, linked to the malignant phenotype observed in Aldrin-exposed cells.
]]>Proteomes doi: 10.3390/proteomes11010010
Authors: Breyer Woodland Aleksandar Necakov Jens R. Coorssen
Integrative top–down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly ‘under-powered’ in terms of proteoform reduction and thus, limit the quality and depth of routine top–down proteomic analyses.
]]>Proteomes doi: 10.3390/proteomes11010009
Authors: Jonathan Munera López Andrés Mariano Alonso Maria Julia Figueras Ana María Saldarriaga Cartagena Miryam A. Hortua Triana Luis Diambra Laura Vanagas Bin Deng Silvia N. J. Moreno Sergio Oscar Angel
Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein–protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.
]]>Proteomes doi: 10.3390/proteomes11010008
Authors: Santosh A. Misal Shital D. Ovhal Sujun Li Jonathan A. Karty Haixu Tang Predrag Radivojac James P. Reilly
Staphylococcus aureus is one of the major community-acquired human pathogens, with growing multidrug-resistance, leading to a major threat of more prevalent infections to humans. A variety of virulence factors and toxic proteins are secreted during infection via the general secretory (Sec) pathway, which requires an N-terminal signal peptide to be cleaved from the N-terminus of the protein. This N-terminal signal peptide is recognized and processed by a type I signal peptidase (SPase). SPase-mediated signal peptide processing is the crucial step in the pathogenicity of S. aureus. In the present study, the SPase-mediated N-terminal protein processing and their cleavage specificity were evaluated using a combination of N-terminal amidination bottom-up and top-down proteomics-based mass spectrometry approaches. Secretory proteins were found to be cleaved by SPase, specifically and non-specifically, on both sides of the normal SPase cleavage site. The non-specific cleavages occur at the relatively smaller residues that are present next to the −1, +1, and +2 locations from the original SPase cleavage site to a lesser extent. Additional random cleavages at the middle and near the C-terminus of some protein sequences were also observed. This additional processing could be a part of some stress conditions and unknown signal peptidase mechanisms.
]]>Proteomes doi: 10.3390/proteomes11010007
Authors: Xian Yu Richard Wilson Alieta Eyles Sadegh Balotf Robert Stephen Tegg Calum Rae Wilson
For potato crops, host resistance is currently the most effective and sustainable tool to manage diseases caused by the plasmodiophorid Spongospora subterranea. Arguably, zoospore root attachment is the most critical phase of infection; however, the underlying mechanisms remain unknown. This study investigated the potential role of root-surface cell-wall polysaccharides and proteins in cultivars resistant/susceptible to zoospore attachment. We first compared the effects of enzymatic removal of root cell-wall proteins, N-linked glycans and polysaccharides on S. subterranea attachment. Subsequent analysis of peptides released by trypsin shaving (TS) of root segments identified 262 proteins that were differentially abundant between cultivars. These were enriched in root-surface-derived peptides but also included intracellular proteins, e.g., proteins associated with glutathione metabolism and lignin biosynthesis, which were more abundant in the resistant cultivar. Comparison with whole-root proteomic analysis of the same cultivars identified 226 proteins specific to the TS dataset, of which 188 were significantly different. Among these, the pathogen-defence-related cell-wall protein stem 28 kDa glycoprotein and two major latex proteins were significantly less abundant in the resistant cultivar. A further major latex protein was reduced in the resistant cultivar in both the TS and whole-root datasets. In contrast, three glutathione S-transferase proteins were more abundant in the resistant cultivar (TS-specific), while the protein glucan endo-1,3-beta-glucosidase was increased in both datasets. These results imply a particular role for major latex proteins and glucan endo-1,3-beta-glucosidase in regulating zoospore binding to potato roots and susceptibility to S. subterranea.
]]>Proteomes doi: 10.3390/proteomes11010006
Authors: Rei Noguchi Akihiro Yoshimura Junji Uchino Takayuki Takeda Yusuke Chihara Takayo Ota Osamu Hiranuma Hiroshi Gyotoku Koichi Takayama Tadashi Kondo
EGFR mutations are strong predictive markers for EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy in patients with non-small-cell lung cancer (NSCLC). Although NSCLC patients with sensitizing EGFR mutations have better prognoses, some patients exhibit worse prognoses. We hypothesized that various activities of kinases could be potential predictive biomarkers for EGFR-TKI treatment among NSCLC patients with sensitizing EGFR mutations. In 18 patients with stage IV NSCLC, EGFR mutations were detected and comprehensive kinase activity profiling was performed using the peptide array PamStation12 for 100 tyrosine kinases. Prognoses were observed prospectively after the administration of EGFR-TKIs. Finally, the kinase profiles were analyzed in combination with the prognoses of the patients. Comprehensive kinase activity analysis identified specific kinase features, consisting of 102 peptides and 35 kinases, in NSCLC patients with sensitizing EGFR mutations. Network analysis revealed seven highly phosphorylated kinases: CTNNB1, CRK, EGFR, ERBB2, PIK3R1, PLCG1, and PTPN11. Pathway analysis and Reactome analysis revealed that the PI3K-AKT and RAF/ MAPK pathways were significantly enriched in the poor prognosis group, being consistent with the outcome of the network analysis. Patients with poor prognoses exhibited high activation of EGFR, PIK3R1, and ERBB2. Comprehensive kinase activity profiles may provide predictive biomarker candidates for screening patients with advanced NSCLC harboring sensitizing EGFR mutations.
]]>Proteomes doi: 10.3390/proteomes11010005
Authors: Kexin Li Qingji Huo Bai-Yan Li Hiroki Yokota
Unlike a prevalent expectation that tumor cells secrete tumor-promoting proteins and stimulate the progression of neighboring tumor cells, accumulating evidence indicates that the role of tumor-secreted proteins is double-edged and context-dependent. Some of the oncogenic proteins in the cytoplasm and cell membranes, which are considered to promote the proliferation and migration of tumor cells, may inversely act as tumor-suppressing proteins in the extracellular domain. Furthermore, the action of tumor-secreted proteins by aggressive “super-fit” tumor cells can be different from those derived from “less-fit” tumor cells. Tumor cells that are exposed to chemotherapeutic agents could alter their secretory proteomes. Super-fit tumor cells tend to secrete tumor-suppressing proteins, while less-fit or chemotherapeutic agent-treated tumor cells may secrete tumor-promotive proteomes. Interestingly, proteomes derived from nontumor cells such as mesenchymal stem cells and peripheral blood mononuclear cells mostly share common features with tumor cell-derived proteomes in response to certain signals. This review introduces the double-sided functions of tumor-secreted proteins and describes the proposed underlying mechanism, which would possibly be based on cell competition.
]]>Proteomes doi: 10.3390/proteomes11010004
Authors: Proteomes Editorial Office Proteomes Editorial Office
High-quality academic publishing is built on rigorous peer review [...]
]]>Proteomes doi: 10.3390/proteomes11010003
Authors: Kathleen A. Heck Håvard T. Lindholm Barbara Niederdorfer Eirini Tsirvouli Martin Kuiper Åsmund Flobak Astrid Lægreid Liv Thommesen
Colorectal cancer (CRC) is one of the most prevalent cancers, driven by several factors including deregulations in intracellular signalling pathways. Small extracellular vesicles (sEVs) are nanosized protein-packaged particles released from cells, which are present in liquid biopsies. Here, we characterised the proteome landscape of sEVs and their cells of origin in three CRC cell lines HCT116, HT29 and SW620 to explore molecular traits that could be exploited as cancer biomarker candidates and how intracellular signalling can be assessed by sEV analysis instead of directly obtaining the cell of origin itself. Our findings revealed that sEV cargo clearly reflects its cell of origin with proteins of the PI3K-AKT pathway highly represented in sEVs. Proteins known to be involved in CRC were detected in both cells and sEVs including KRAS, ARAF, mTOR, PDPK1 and MAPK1, while TGFB1 and TGFBR2, known to be key players in epithelial cancer carcinogenesis, were found to be enriched in sEVs. Furthermore, the phosphopeptide-enriched profiling of cell lysates demonstrated a distinct pattern between cell lines and highlighted potential phosphoproteomic targets to be investigated in sEVs. The total proteomic and phosphoproteomics profiles described in the current work can serve as a source to identify candidates for cancer biomarkers that can potentially be assessed from liquid biopsies.
]]>Proteomes doi: 10.3390/proteomes11010002
Authors: Ankita Punetha Deepak Kotiya
Proteomics continues to forge significant strides in the discovery of essential biological processes, uncovering valuable information on the identity, global protein abundance, protein modifications, proteoform levels, and signal transduction pathways. Cancer is a complicated and heterogeneous disease, and the onset and progression involve multiple dysregulated proteoforms and their downstream signaling pathways. These are modulated by various factors such as molecular, genetic, tissue, cellular, ethnic/racial, socioeconomic status, environmental, and demographic differences that vary with time. The knowledge of cancer has improved the treatment and clinical management; however, the survival rates have not increased significantly, and cancer remains a major cause of mortality. Oncoproteomics studies help to develop and validate proteomics technologies for routine application in clinical laboratories for (1) diagnostic and prognostic categorization of cancer, (2) real-time monitoring of treatment, (3) assessing drug efficacy and toxicity, (4) therapeutic modulations based on the changes with prognosis and drug resistance, and (5) personalized medication. Investigation of tumor-specific proteomic profiles in conjunction with healthy controls provides crucial information in mechanistic studies on tumorigenesis, metastasis, and drug resistance. This review provides an overview of proteomics technologies that assist the discovery of novel drug targets, biomarkers for early detection, surveillance, prognosis, drug monitoring, and tailoring therapy to the cancer patient. The information gained from such technologies has drastically improved cancer research. We further provide exemplars from recent oncoproteomics applications in the discovery of biomarkers in various cancers, drug discovery, and clinical treatment. Overall, the future of oncoproteomics holds enormous potential for translating technologies from the bench to the bedside.
]]>Proteomes doi: 10.3390/proteomes11010001
Authors: Francesca Vignaroli Angelica Mele Giacomo Tondo Veronica De Giorgis Marcello Manfredi Cristoforo Comi Letizia Mazzini Fabiola De Marchi
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severely debilitating and progressive neurodegenerative disorders. A distinctive pathological feature of several neurodegenerative diseases, including ALS and FTD, is the deposition of aberrant protein inclusions in neuronal cells, which leads to cellular dysfunction and neuronal damage and loss. Despite this, to date, the biological process behind developing these protein inclusions must be better clarified, making the development of disease-modifying treatment impossible until this is done. Proteomics is a powerful tool to characterize the expression, structure, functions, interactions, and modifications of proteins of tissue and biological fluid, including plasma, serum, and cerebrospinal fluid. This protein-profiling characterization aims to identify disease-specific protein alteration or specific pathology-based mechanisms which may be used as markers of these conditions. Our narrative review aims to highlight the need for biomarkers and the potential use of proteomics in clinical practice for ALS–FTD spectrum disorders, considering the emerging rationale in proteomics for new drug development. Certainly, new data will emerge in the near future in this regard and support clinicians in the development of personalized medicine.
]]>Proteomes doi: 10.3390/proteomes10040039
Authors: Jens Möller Mona Bodenschatz Vartul Sangal Jörg Hofmann Andreas Burkovski
Corynebacterium pseudotuberculosis is an important animal pathogen, which is also able to infect humans. An optimal treatment of infections with this pathogen is not available today and consequently, more research is necessary to understand the infection process. Here, we present a combined -omics and bioinformatics approach to characterize C. pseudotuberculosis 12CS0282. The genome sequence of strain 12CS0282 was determined, analyzed in comparison with the available 130 C. pseudotuberculosis sequences and used as a basis for proteome analyses. In a reverse vaccinology approach, putative vaccine and drug targets for 12CS0208 were identified. Mass spectrometry analyses revealed the presence of multiple virulence factors even without host contact. In macrophage interaction studies, C. pseudotuberculosis 12CS0282 was highly resistant against human phagocytes and even multiplied within human THP-1 cells. Taken together, the data indicate a high pathogenic potential of the strain.
]]>Proteomes doi: 10.3390/proteomes10040038
Authors: Ramesh Kumar Divya Mehta Sakshi Chaudhary Debasis Nayak Sujatha Sunil
Arboviruses are some of the important causative agents of mosquito-mediated viral diseases. These viruses are transmitted between vector and host during the blood meal. Upon viral entry, host replication machinery is hijacked, supporting new virus particle production and thereby allowing viral survival in the host. In this process, host proteins interact with viral proteins to either facilitate viral replication, or they may provide antiviral defense mechanisms. In this study, we analyzed the impact of chikungunya virus (CHIKV) infection on the global proteome of Dicer active Aedes albopictus cells during the early and late time points of infection. We utilized a bottom-up approach of global proteomics analysis, and we used label-free quantitative mass spectrometry to identify the global protein signatures of Ae. albopictus at two different time points upon CHIKV infection. The mass spectrometry data analysis of the early time point revealed that proteins belonging to pathways such as translation, RNA processing, and cellular metabolic processes were less in abundance, whereas those belonging to pathways such as cellular catabolic process and organic substance transport were significantly abundant. At later time points, proteins belonging to pathways such as cellular metabolic processes, primary metabolic process, organonitrogen compound metabolic process, and organic substance metabolic process were found to be decreased in their presence, whereas those belonging to pathways such as RNA processing, gene expression, macromolecule metabolic processing, and nitrogen compound metabolic processing were found to be abundant during CHIKV infection, indicating that modulation in gene expression favoring cell survival occurs at a later time point, suggesting a survival strategy of Aedes cells to counter prolonged CHIKV infection.
]]>Proteomes doi: 10.3390/proteomes10040037
Authors: Gabriele Riccardi Mario Giuseppe Bellizzi Irene Fatuzzo Federica Zoccali Luca Cavalcanti Antonio Greco Marco de Vincentiis Massimo Ralli Marco Fiore Carla Petrella Antonio Minni Christian Barbato
Background: Oral squamous cell carcinoma (OSCC) is one of the most frequent cancers worldwide. Endoscopic methods may be useful in the evaluation of oral injuries even though the diagnostic gold standard is a biopsy. Targeted screenings could be considered the best way to prevent the occurrence of oral cancer. Aimed to elucidate the potential identification of specific biomarkers of OSCC, the use of saliva is convenient and noninvasive. Many studies reported more than a hundred putative saliva biomarkers for OSCC, and proteogenomic approaches were fundamental to disclosing this issue. Methods: Relevant literature published in the last few years was systematically searched on PubMed and we focused on articles about the use and study of salivary biomarkers in the diagnostics of head and neck cancer (n = 110). Thereafter, we performed a selection focusing on diagnosis with salivary proteomics in OSCC (n = 8). Results: Saliva proteomics can be a source of biomarkers for OSCC. We reviewed literature of biomarker proteins in saliva that could also be evaluated as probable targets for non-invasive screening of oral neoplasm such as cytokines, matrix metalloproteinases, and acute-phase response proteins. Conclusions: The measurement of salivary biomarkers is a highly hopeful technique for the diagnosis of OSCC. Proteogenomic approaches could permit an accurate and early diagnosis of OSCC. This review seeks to generate an up-to-date view on translational OSCC issues by raising awareness of researchers, physicians, and surgeons. Renewed clinical studies, which will validate the sensitivity and specificity of salivary biomarkers, are necessary to translate these results into possible strategies for early diagnosis of OSCC, thus improving patient outcomes.
]]>Proteomes doi: 10.3390/proteomes10040036
Authors: Roshanak Aslebagh Danielle Whitham Devika Channaveerappa Panashe Mutsengi Brian T. Pentecost Kathleen F. Arcaro Costel C. Darie
It is thought that accurate risk assessment and early diagnosis of breast cancer (BC) can help reduce cancer-related mortality. Proteomics analysis of breast milk may provide biomarkers of risk and occult disease. Our group works on the analysis of human milk samples from women with BC and controls to investigate alterations in protein patterns of milk that could be related to BC. In the current study, we used mass spectrometry (MS)-based proteomics analysis of 12 milk samples from donors with BC and matched controls. Specifically, we used one-dimensional (1D)-polyacrylamide gel electrophoresis (PAGE) coupled with nanoliquid chromatography tandem MS (nanoLC-MS/MS), followed by bioinformatics analysis. We confirmed the dysregulation of several proteins identified previously in a different set of milk samples. We also identified additional dysregulations in milk proteins shown to play a role in cancer development, such as Lactadherin isoform A, O-linked N-acetylglucosamine (GlcNAc) transferase, galactosyltransferase, recoverin, perilipin-3 isoform 1, histone-lysine methyltransferase, or clathrin heavy chain. Our results expand our current understanding of using milk as a biological fluid for identification of BC-related dysregulated proteins. Overall, our results also indicate that milk has the potential to be used for BC biomarker discovery, early detection and risk assessment in young, reproductively active women.
]]>Proteomes doi: 10.3390/proteomes10040035
Authors: Anca-Narcisa Neagu Madhuri Jayathirtha Danielle Whitham Panashe Mutsengi Isabelle Sullivan Brindusa Alina Petre Costel C. Darie
Immunohistochemistry (IHC) is still widely used as a morphology-based assay for in situ analysis of target proteins as specific tumor antigens. However, as a very heterogeneous collection of neoplastic diseases, breast cancer (BC) requires an accurate identification and characterization of larger panels of candidate biomarkers, beyond ER, PR, and HER2 proteins, for diagnosis and personalized treatment, without the limited availability of antibodies that are required to identify specific proteins. Top-down, middle-down, and bottom-up mass spectrometry (MS)-based proteomics approaches complement traditional histopathological tissue analysis to examine expression, modification, and interaction of hundreds to thousands of proteins simultaneously. In this review, we discuss the proteomics-based identification of dysregulated proteins in BC that are essential for the following issues: discovery and validation of new biomarkers by analysis of solid and liquid/non-invasive biopsies, cell lines, organoids and xenograft models; identification of panels of biomarkers for early detection and accurate discrimination between cancer, benign and normal tissues; identification of subtype-specific and stage-specific protein expression profiles in BC grading and measurement of disease progression; characterization of new subtypes of BC; characterization and quantitation of post-translational modifications (PTMs) and aberrant protein–protein interactions (PPI) involved in tumor development; characterization of the global remodeling of BC tissue homeostasis, diagnosis and prognostic information; and deciphering of molecular functions, biological processes and mechanisms through which the dysregulated proteins cause tumor initiation, invasion, and treatment resistance.
]]>Proteomes doi: 10.3390/proteomes10040034
Authors: Mukul Jain Nil Patil Darshil Gor Mohit Kumar Sharma Neha Goel Prashant Kaushik
The novel SARS-CoV-2 variant, Omicron (B.1.1.529), is being testified, and the WHO has characterized Omicron as a variant of concern due to its higher transmissibility and very contagious behavior, immunization breakthrough cases. Here, the comparative proteomic study has been conducted on spike-protein, hACE2 of five lineages (α, β, δ, γ and Omicron. The docking was performed on spike protein- hACE-2 protein using HADDOCK, and PRODIGY was used to analyze the binding energy affinity using a reduced Haddock score. Followed by superimposition in different variant-based protein structures and calculated the esteem root mean square deviation (RMSD). This study reveals that Omicron was seen generating a monophyletic clade. Further, as α variant is the principal advanced strain after Wuhan SARS-CoV-2, and that is the reason it was showing the least likeness rate with the Omicron and connoting Omicron has developed of late with the extreme number of mutations. α variant has shown the highest binding affinity with hACE2, followed by β strain, and followed with γ. Omicron showed a penultimate binding relationship, while the δ variant was seen as having the least binding affinity. This proteomic basis in silico analysis of variable spike proteins of variants will impart light on the development of vaccines and the identification of mutations occurring in the upcoming variants.
]]>Proteomes doi: 10.3390/proteomes10030033
Authors: Matthew B. O’Rourke Ben R. Roediger Christopher J. Jolly Ben Crossett Matthew P. Padula Phillip M. Hansbro
(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.
]]>Proteomes doi: 10.3390/proteomes10030032
Authors: Nadeeka Thushari Gajahin Gamage Rina Miyashita Kazutaka Takahashi Shuichi Asakawa Jayan Duminda Mahesh Senevirathna
Genome determines the unique individualities of organisms; however, proteins play significant roles in the generation of the colorful life forms below water. Aquatic systems are usually complex and multifaceted and can take on unique modifications and adaptations to environmental changes by altering proteins at the cellular level. Proteomics is an essential strategy for exploring aquatic ecosystems due to the diverse involvement of proteins, proteoforms, and their complexity in basic and advanced cellular functions. Proteomics can expedite the analysis of molecular mechanisms underlying biological processes in an aquatic environment. Previous proteomic studies on aquatic environments have mainly focused on pollution assessments, ecotoxicology, their role in the food industry, and extraction and identification of natural products. Aquatic protein biomarkers have been comprehensively reported and are currently extensively applied in the pharmaceutical and medical industries. Cellular- and molecular-level responses of organisms can be used as indicators of environmental changes and stresses. Conversely, environmental changes are expedient in predicting aquatic health and productivity, which are crucial for ecosystem management and conservation. Recent advances in proteomics have contributed to the development of sustainable aquaculture, seafood safety, and high aquatic food production. Proteomic approaches have expanded to other aspects of the aquatic environment, such as protein fingerprinting for species identification. In this review, we encapsulated current proteomic applications and evaluated the potential strengths, weaknesses, opportunities, and threats of proteomics for future aquatic environmental studies. The review identifies both pros and cons of aquatic proteomics and projects potential challenges and recommendations. We postulate that proteomics is an emerging, powerful, and integrated omics approach for aquatic environmental studies.
]]>Proteomes doi: 10.3390/proteomes10030031
Authors: A. D. A. Shahinuzzaman Abu Hena Mostafa Kamal Jayanta K. Chakrabarty Aurchie Rahman Saiful M. Chowdhury
Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4.
]]>Proteomes doi: 10.3390/proteomes10030030
Authors: Fábio Trindade Ana F. Ferreira Francisca Saraiva Diana Martins Vera M. Mendes Carla Sousa Cristina Gavina Adelino Leite-Moreira Bruno Manadas Inês Falcão-Pires Rui Vitorino
The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex.
]]>Proteomes doi: 10.3390/proteomes10030029
Authors: Diego Fernández-Lázaro Evelina Garrosa Jesús Seco-Calvo Manuel Garrosa
Sarcopenia (Sp) is the loss of skeletal muscle mass associated with aging which causes an involution of muscle function and strength. Satellite cells (Sc) are myogenic stem cells, which are activated by injury or stress, and repair muscle tissue. With advancing age, there is a decrease in the efficiency of the regenerative response of Sc. Diagnosis occurs with the Sp established by direct assessments of muscle. However, the detection of biomarkers in real-time biofluids by liquid biopsy could represent a step-change in the understanding of the molecular biology and heterogeneity of Sp. A total of 13 potential proteogenomic biomarkers of Sp by their physiological and biological interaction with Sc have been previously described in the literature. Increases in the expression of GDF11, PGC-1α, Sirt1, Pax7, Pax3, Myf5, MyoD, CD34, MyoG, and activation of Notch signaling stimulate Sc activity and proliferation, which could modulate and delay Sp progression. On the contrary, intensified expression of GDF8, p16INK4a, Mrf4, and activation of the Wnt pathway would contribute to early Sp development by directly inducing reduced and/or altered Sc function, which would attenuate the restorative capacity of skeletal muscle. Additionally, tissue biopsy remains an important diagnostic tool. Proteomic profiling of aged muscle tissues has shown shifts toward protein isoforms characteristic of a fast-to-slow transition process and an elevated number of oxidized proteins. In addition, a strong association between age and plasma values of growth differentiation factor 15 (GDF-15) has been described and serpin family A member 3 (serpin A3n) was more secreted by atrophied muscle cells. The identification of these new biomarkers holds the potential to change personalized medicine because it could predict in real time the course of Sp by monitoring its evolution and assessing responses to potential therapeutic strategies.
]]>Proteomes doi: 10.3390/proteomes10030028
Authors: Timo Längrich Kaya Bork Rüdiger Horstkorte Veronika Weber Britt Hofmann Matt Fuszard Heidi Olzscha
Background: Propofol is a short-acting anesthetic, which is often used for induction and maintenance of general anesthesia, sedation for mechanically ventilated adults and procedural sedation. Several side effects of propofol are known and a substantial number of patients suffer from post-operative delirium after propofol application. In this study, we analyzed the effect of propofol on the function and protein expression profile on a proteome-wide scale. Methods: We cultured human brain microvascular endothelial cells in absence and presence of propofol and analyzed the permeability of the blood-brain barrier (BBB) by fluorescein passage and protein abundance on a proteome-wide scale by mass spectrometry. Results: Propofol interfered with the function of the blood-brain barrier. This was not due to decreased adhesion of propofol-treated human brain microvascular endothelial cells. The proteomic analysis revealed that some key pathways in these cells were disturbed, such as oxygen metabolism, DNA damage recognition and response to stress. Conclusions: Propofol has strong effects on protein expression which could explain several side effects of propofol.
]]>Proteomes doi: 10.3390/proteomes10030027
Authors: Tindaro Bongiovanni Mathieu Lacome Vassilios Fanos Giulia Martera Erika Cione Roberto Cannataro
Metabolomics is a promising tool for studying exercise physiology and exercise-associated metabolism. It has recently been defined with the term “sportomics” due to metabolomics’ capability to characterize several metabolites in several biological samples simultaneously. This narrative review on exercise metabolomics provides an initial and brief overview of the different metabolomics technologies, sample collection, and further processing steps employed for sport. It also discusses the data analysis and its biological interpretation. Thus, we do not cover sample collection, preparation, and analysis paragraphs in detail here but outline a general outlook to help the reader to understand the metabolomics studies conducted in team-sports athletes, alongside endeavoring to recognize existing or emergent trends and deal with upcoming directions in the field of exercise metabolomics in a team-sports setting.
]]>Proteomes doi: 10.3390/proteomes10030026
Authors: Shivangi Awasthi Daniel S. Spellman Nathan G. Hatcher
Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral β amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.
]]>Proteomes doi: 10.3390/proteomes10030025
Authors: Donatella Pia Spanò Simone Bonelli Matteo Calligaris Anna Paola Carreca Claudia Carcione Giovanni Zito Aldo Nicosia Sergio Rizzo Simone Dario Scilabra
Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.
]]>Proteomes doi: 10.3390/proteomes10030024
Authors: Luís M. Ramalhete Rúben Araújo Aníbal Ferreira Cecília R. C. Calado
Renal transplantation is currently the treatment of choice for end-stage kidney disease, enabling a quality of life superior to dialysis. Despite this, all transplanted patients are at risk of allograft rejection processes. The gold-standard diagnosis of graft rejection, based on histological analysis of kidney biopsy, is prone to sampling errors and carries high costs and risks associated with such invasive procedures. Furthermore, the routine clinical monitoring, based on urine volume, proteinuria, and serum creatinine, usually only detects alterations after graft histologic damage and does not differentiate between the diverse etiologies. Therefore, there is an urgent need for new biomarkers enabling to predict, with high sensitivity and specificity, the rejection processes and the underlying mechanisms obtained from minimally invasive procedures to be implemented in routine clinical surveillance. These new biomarkers should also detect the rejection processes as early as possible, ideally before the 78 clinical outputs, while enabling balanced immunotherapy in order to minimize rejections and reducing the high toxicities associated with these drugs. Proteomics of biofluids, collected through non-invasive or minimally invasive analysis, e.g., blood or urine, present inherent characteristics that may provide biomarker candidates. The current manuscript reviews biofluids proteomics toward biomarkers discovery that specifically identify subclinical, acute, and chronic immune rejection processes while allowing for the discrimination between cell-mediated or antibody-mediated processes. In time, these biomarkers will lead to patient risk stratification, monitoring, and personalized and more efficient immunotherapies toward higher graft survival and patient quality of life.
]]>Proteomes doi: 10.3390/proteomes10030023
Authors: Ang Luo Rongrong Hao Xia Zhou Yangfan Jia Haiyang Tang
Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the main causes of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Hypoxia is an important factor related to PAH and can induce the excessive proliferation of PASMCs and inhibit apoptosis. To explore the possible mechanism of hypoxia-related PAH, human PASMCs are exposed to hypoxia for 24 h and tandem mass tag (TMT)-based quantitative proteomic and phosphoproteomic analyses are performed. Proteomic analysis revealed 134 proteins are significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]), of which 48 proteins are upregulated and 86 are downregulated. Some of the changed proteins are verified by using qRT-PCR and Western blotting. Phosphoproteomic analysis identified 404 significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]) phosphopeptides. Among them, 146 peptides are upregulated while 258 ones are downregulated. The kinase-substrate enrichment analysis revealed kinases such as P21 protein-activated kinase 1/2/4 (PAK1/2/4), protein-kinase cGMP-dependent 1 and 2 (PRKG1/2), and mitogen-activated protein-kinase 4/6/7 (MAP2K4/6/7) are significantly enriched and activated. For all the significantly changed proteins or phosphoproteins, a comprehensive pathway analysis is performed. In general, this study furthers our understanding of the mechanism of hypoxia-induced PAH.
]]>Proteomes doi: 10.3390/proteomes10020022
Authors: Joel R. Steele Carly J. Italiano Connor R. Phillips Jake P. Violi Lisa Pu Kenneth J. Rodgers Matthew P. Padula
In the original publication, there was a mistake in Table 2 as published [...]
]]>Proteomes doi: 10.3390/proteomes10020021
Authors: Adam J. O’Neal Katherine A. Glass Christopher J. Emig Adela A. Vitug Steven J. Henry Dikoma C. Shungu Xiangling Mao Susan M. Levine Maureen R. Hanson
Infectious pathogens are implicated in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) because of the occurrence of outbreaks of the disease. While a number of different infectious agents have been associated with the onset of ME/CFS, the identity of a specific organism has been difficult to determine in individual cases. The aim of our study is to survey ME/CFS subjects for evidence of an infectious trigger and/or evidence of immune dysregulation via serological testing of plasma samples for antibodies to 122 different pathogen antigens. Immune profiles were compared to age-, sex-, and BMI-matched controls to provide a basis for comparison. Antibody levels to individual antigens surveyed in this study do not implicate any one of the pathogens in ME/CFS, nor do they rule out common pathogens that frequently infect the US population. However, our results revealed sex-based differences in steady-state humoral immunity, both within the ME/CFS cohort and when compared to trends seen in the healthy control cohort.
]]>Proteomes doi: 10.3390/proteomes10020020
Authors: Sarah Jane Annesley Claire Yvonne Allan Oana Sanislav Andrew Evans Paul Robert Fisher
Parkinson’s disease is the second largest neurodegenerative disease worldwide and is caused by a combination of genetics and environment. It is characterized by the death of neurons in the substantia nigra of the brain but is not solely a disease of the brain, as it affects multiple tissues and organs. Studying Parkinson’s disease in accessible tissues such as skin and blood has increased our understanding of the disease’s pathogenesis. Here, we used lymphoblast cell lines generated from Parkinson’s disease patient and healthy age- and sex-matched control groups and obtained their whole-cell transcriptomes and proteomes. Our analysis revealed, in both the transcriptomes and the proteomes of PD cells, a global downregulation of genes involved in protein synthesis, as well as the upregulation of immune processes and sphingolipid metabolism. In contrast, we discovered an uncoupling of mRNA and protein expression in processes associated with mitochondrial respiration in the form of a general downregulation in associated transcripts and an upregulation in proteins. Complex V was different to the other oxidative phosphorylation complexes in that the levels of its associated transcripts were also lower, but the levels of their encoded polypeptides were not elevated. This may suggest that further layers of regulation specific to Complex V are in play.
]]>Proteomes doi: 10.3390/proteomes10020019
Authors: Natalie P. Turner Pevindu Abeysinghe Keith A. Kwan Cheung Kanchan Vaswani Jayden Logan Pawel Sadowski Murray D. Mitchell
Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A ‘gold-standard’ method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.
]]>Proteomes doi: 10.3390/proteomes10020018
Authors: Jordan B. Burton Nicholas J. Carruthers Zhanjun Hou Larry H. Matherly Paul M. Stemmer
Localization of organelle proteins by isotope tagging (LOPIT) maps are a coordinate-directed representation of proteome data that can aid in biological interpretation. Analysis of organellar association for proteins as displayed using LOPIT is evaluated and interpreted for two types of proteomic data sets. First, test and control group protein abundances and fold change data obtained in a proximity labeling experiment are plotted on a LOPIT map to evaluate the likelihood of true protein interactions. Selection of true positives based on co-localization of proteins in the organellar space is shown to be consistent with carboxylase enrichment which serves as a positive control for biotinylation in streptavidin affinity selected proteome data sets. The mapping in organellar space facilitates discrimination between the test and control groups and aids in identification of proteins of interest. The same representation of proteins in organellar space is used in the analysis of extracellular vesicle proteomes for which protein abundance and fold change data are evaluated. Vesicular protein organellar localization patterns provide information about the subcellular origin of the proteins in the samples which are isolates from the extracellular milieu. The organellar localization patterns are indicative of the provenance of the vesicular proteome origin and allow discrimination between proteomes prepared using different enrichment methods. The patterns in LOPIT displays are easy to understand and compare which aids in the biological interpretation of proteome data.
]]>Proteomes doi: 10.3390/proteomes10020017
Authors: Tanushree Halder Mukesh Choudhary Hui Liu Yinglong Chen Guijun Yan Kadambot H. M. Siddique
Wheat is an important staple cereal for global food security. However, climate change is hampering wheat production due to abiotic stresses, such as heat, salinity, and drought. Besides shoot architectural traits, improving root system architecture (RSA) traits have the potential to improve yields under normal and stressed environments. RSA growth and development and other stress responses involve the expression of proteins encoded by the trait controlling gene/genes. Hence, mining the key proteins associated with abiotic stress responses and RSA is important for improving sustainable yields in wheat. Proteomic studies in wheat started in the early 21st century using the two-dimensional (2-DE) gel technique and have extensively improved over time with advancements in mass spectrometry. The availability of the wheat reference genome has allowed the exploration of proteomics to identify differentially expressed or abundant proteins (DEPs or DAPs) for abiotic stress tolerance and RSA improvement. Proteomics contributed significantly to identifying key proteins imparting abiotic stress tolerance, primarily related to photosynthesis, protein synthesis, carbon metabolism, redox homeostasis, defense response, energy metabolism and signal transduction. However, the use of proteomics to improve RSA traits in wheat is in its infancy. Proteins related to cell wall biogenesis, carbohydrate metabolism, brassinosteroid biosynthesis, and transportation are involved in the growth and development of several RSA traits. This review covers advances in quantification techniques of proteomics, progress in identifying DEPs and/or DAPs for heat, salinity, and drought stresses, and RSA traits, and the limitations and future directions for harnessing proteomics in wheat improvement.
]]>Proteomes doi: 10.3390/proteomes10020016
Authors: Aruni Ghose Sri Vidya Niharika Gullapalli Naila Chohan Anita Bolina Michele Moschetta Elie Rassy Stergios Boussios
The ability to identify ovarian cancer (OC) at its earliest stages remains a challenge. The patients present an advanced stage at diagnosis. This heterogeneous disease has distinguishable etiology and molecular biology. Next-generation sequencing changed clinical diagnostic testing, allowing assessment of multiple genes, simultaneously, in a faster and cheaper manner than sequential single gene analysis. Technologies of proteomics, such as mass spectrometry (MS) and protein array analysis, have advanced the dissection of the underlying molecular signaling events and the proteomic characterization of OC. Proteomics analysis of OC, as well as their adaptive responses to therapy, can uncover new therapeutic choices, which can reduce the emergence of drug resistance and potentially improve patient outcomes. There is an urgent need to better understand how the genomic and epigenomic heterogeneity intrinsic to OC is reflected at the protein level, and how this information could potentially lead to prolonged survival.
]]>Proteomes doi: 10.3390/proteomes10020015
Authors: Sisse Andersen Arkadiusz Nawrocki Andreas Eske Johansen Ana Herrero-Fresno Vanesa García Menéndez Jakob Møller-Jensen John Elmerdahl Olsen
Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, they undergo metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. To understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of the UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, most prominently arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were likewise upregulated together with proteins involved in sulfur compound turnover. Ribosomal proteins were downregulated relative to growth in MOPS in this environment. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.
]]>Proteomes doi: 10.3390/proteomes10020014
Authors: Vikrant Rai Devendra K. Agrawal
Arteriovenous fistulas (AVFs), created for hemodialysis in end-stage renal disease patients, mature through the outward remodeling of the outflow vein. However, early thrombosis and chronic inflammation are detrimental to the process of AVF maturation and precipitate AVF maturation failure. For the successful remodeling of the outflow vein, blood flow through the fistula is essential, but early arterial thrombosis attenuates this blood flow, and the vessels become thrombosed and stenosed, leading to AVF failure. The altered expression of various proteins involved in maintaining vessel patency or thrombosis is regulated by genes of which the expression is regulated by transcription factors and microRNAs. In this study, using thrombosed and stenosed arteries following AVF creation, we delineated transcription factors and microRNAs associated with differentially expressed genes in bulk RNA sequencing data using upstream and causal network analysis. We observed changes in many transcription factors and microRNAs that are involved in angiogenesis; vascular smooth muscle cell proliferation, migration, and phenotypic changes; endothelial cell function; hypoxia; oxidative stress; vessel remodeling; immune responses; and inflammation. These factors and microRNAs play a critical role in the underlying molecular mechanisms in AVF maturation. We also observed epigenetic factors involved in gene regulation associated with these molecular mechanisms. The results of this study indicate the importance of investigating the transcriptional and epigenetic regulation of AVF maturation and maturation failure and targeting factors precipitating early thrombosis and stenosis.
]]>Proteomes doi: 10.3390/proteomes10020013
Authors: Amrita Mukherjee Chinmayi Bhagwan Pednekar Siddhant Sujit Kolke Megha Kattimani Subhiksha Duraisamy Ananya Raghu Burli Sudeep Gupta Sanjeeva Srivastava
Cervical cancer is one of the top malignancies in women around the globe, which still holds its place despite being preventable at early stages. Gynecological conditions, even maladies like cervical cancer, still experience scrutiny from society owing to prevalent taboo and invasive screening methods, especially in developing economies. Additionally, current diagnoses lack specificity and sensitivity, which prolong diagnosis until it is too late. Advances in omics-based technologies aid in discovering differential multi-omics profiles between healthy individuals and cancer patients, which could be utilized for the discovery of body fluid-based biomarkers. Body fluids are a promising potential alternative for early disease detection and counteracting the problems of invasiveness while also serving as a pool of potential biomarkers. In this review, we will provide details of the body fluids-based biomarkers that have been reported in cervical cancer. Here, we have presented our perspective on proteomics for global biomarker discovery by addressing several pertinent problems, including the challenges that are confronted in cervical cancer. Further, we also used bioinformatic methods to undertake a meta-analysis of significantly up-regulated biomolecular profiles in CVF from cervical cancer patients. Our analysis deciphered alterations in the biological pathways in CVF such as immune response, glycolytic processes, regulation of cell death, regulation of structural size, protein polymerization disease, and other pathways that can cumulatively contribute to cervical cancer malignancy. We believe, more extensive research on such biomarkers, will speed up the road to early identification and prevention of cervical cancer in the near future.
]]>Proteomes doi: 10.3390/proteomes10020012
Authors: Enxhi Shaba Lorenza Vantaggiato Laura Governini Alesandro Haxhiu Guido Sebastiani Daniela Fignani Giuseppina Emanuela Grieco Laura Bergantini Luca Bini Claudia Landi
In the era of multi-omic sciences, dogma on singular cause-effect in physio-pathological processes is overcome and system biology approaches have been providing new perspectives to see through. In this context, extracellular vesicles (EVs) are offering a new level of complexity, given their role in cellular communication and their activity as mediators of specific signals to target cells or tissues. Indeed, their heterogeneity in terms of content, function, origin and potentiality contribute to the cross-interaction of almost every molecular process occurring in a complex system. Such features make EVs proper biological systems being, therefore, optimal targets of omic sciences. Currently, most studies focus on dissecting EVs content in order to either characterize it or to explore its role in various pathogenic processes at transcriptomic, proteomic, metabolomic, lipidomic and genomic levels. Despite valuable results being provided by individual omic studies, the categorization of EVs biological data might represent a limit to be overcome. For this reason, a multi-omic integrative approach might contribute to explore EVs function, their tissue-specific origin and their potentiality. This review summarizes the state-of-the-art of EVs omic studies, addressing recent research on the integration of EVs multi-level biological data and challenging developments in EVs origin.
]]>Proteomes doi: 10.3390/proteomes10020011
Authors: Andrew T. Rajczewski Qiyuan Han Subina Mehta Praveen Kumar Pratik D. Jagtap Charles G. Knutson James G. Fox Natalia Y. Tretyakova Timothy J. Griffin
Chronic inflammation of the colon causes genomic and/or transcriptomic events, which can lead to expression of non-canonical protein sequences contributing to oncogenesis. To better understand these mechanisms, Rag2−/−Il10−/− mice were infected with Helicobacter hepaticus to induce chronic inflammation of the cecum and the colon. Transcriptomic data from harvested proximal colon samples were used to generate a customized FASTA database containing non-canonical protein sequences. Using a proteogenomic approach, mass spectrometry data for proximal colon proteins were searched against this custom FASTA database using the Galaxy for Proteomics (Galaxy-P) platform. In addition to the increased abundance in inflammatory response proteins, we also discovered several non-canonical peptide sequences derived from unique proteoforms. We confirmed the veracity of these novel sequences using an automated bioinformatics verification workflow with targeted MS-based assays for peptide validation. Our bioinformatics discovery workflow identified 235 putative non-canonical peptide sequences, of which 58 were verified with high confidence and 39 were validated in targeted proteomics assays. This study provides insights into challenges faced when identifying non-canonical peptides using a proteogenomics approach and demonstrates an integrated workflow addressing these challenges. Our bioinformatic discovery and verification workflow is publicly available and accessible via the Galaxy platform and should be valuable in non-canonical peptide identification using proteogenomics.
]]>Proteomes doi: 10.3390/proteomes10020010
Authors: Leonardo Saboia-Vahia Patricia Cuervo Jacek R. Wiśniewski Geovane Dias-Lopes Nathalia Pinho Gabriel Padrón Fernando de Pilla Varotti Silvane Maria Fonseca Murta
Visceral leishmaniasis (VL) is a neglected disease caused by Leishmania parasites. Although significant morbidity and mortality in tropical and subtropical regions of the world are associated with VL, the low investment for developing new treatment measures is chronic. Moreover, resistance and treatment failure are increasing for the main medications, but the emergence of resistance phenotypes is poorly understood at the protein level. Here, we analyzed the development of resistance to miltefosine upon experimental selection in a L. infantum strain. Time to miltefosine resistance emergence was ~six months and label-free quantitative mass-spectrometry-based proteomics analyses revealed that this process involves a remodeling of components of the membrane and mitochondrion, with significant increase in oxidative phosphorylation complexes, particularly on complex IV and ATP synthase, accompanied by increased energy metabolism mainly dependent on β-oxidation of fatty acids. Proteins canonically involved in ROS detoxification did not contribute to the resistant process whereas sterol biosynthesis enzymes could have a role in this development. Furthermore, changes in the abundance of proteins known to be involved in miltefosine resistance such as ABC transporters and phospholipid transport ATPase were detected. Together, our data show a more complete picture of the elements that make up the miltefosine resistance phenotype in L. infantum.
]]>Proteomes doi: 10.3390/proteomes10010009
Authors: Halley Gora Ravuri Zainab Noor Paul C. Mills Nana Satake Pawel Sadowski
Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species.
]]>Proteomes doi: 10.3390/proteomes10010008
Authors: Amir Taldaev Vladimir Rudnev Liudmila Kulikova Kirill Nikolsky Alexander Efimov Kristina Malsagova Anna Kaysheva
Biological activity regulation by protein post-translational modification (PTM) is critical for cell function, development, differentiation, and survival. Dysregulation of PTM proteins is present in various pathological conditions, including rheumatoid arthritis (RA). RA is a systemic autoimmune disease that primarily affects joints, and there are three main types of protein PTMs associated with the development of this disease, namely, glycosylation, citrullination, and carbamylation. Glycosylation is important for the processing and presentation of antigen fragments on the cell surface and can modulate immunoglobulin activity. The citrullination of autoantigens is closely associated with RA, as evidenced by the presence of antibodies specific to citrullinated proteins in the serum of patients. Carbamylation and dysregulation have recently been associated with RA development in humans.In this study, we performed an overview analysis of proteins with post-translational modifications associated with the development of RA adverted in peer-reviewed scientific papers for the past 20 years. As a result of the search, a list of target proteins and corresponding amino acid sequences with PTM in RA was formed. Structural characteristics of the listed modified proteins were extracted from the Protein Data Bank. Then, molecular dynamics experiments of intact protein structures and corresponding structures with PTMs were performed regarding structures in the list announced in the ProtDB service. This study aimed to conduct a molecular dynamics study of intact proteins and proteins, including post-translational modification and protein citrullination, likely associated with RA development. We observed another exhibition of the fundamental physics concept, symmetry, at the submolecular level, unveiled as the autonomous repetitions of outside the protein structural motif performance globule corresponding to those in the whole protein molecule.
]]>Proteomes doi: 10.3390/proteomes10010007
Authors: Kristina J. H. Kleinwort Roxane L. Degroote Sieglinde Hirmer Lucia Korbonits Lea Lorenz Armin M. Scholz Stefanie M. Hauck Cornelia A. Deeg
We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation.
]]>Proteomes doi: 10.3390/proteomes10010006
Authors: Proteomes Editorial Office Proteomes Editorial Office
Rigorous peer-reviews are the basis of high-quality academic publishing [...]
]]>Proteomes doi: 10.3390/proteomes10010005
Authors: Sadegh Balotf Richard Wilson Robert S. Tegg David S. Nichols Calum R. Wilson
The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant–pathogen interaction. Mass spectrometry (MS)-based proteomics has become the preferred method for characterizing proteins at the proteome and sub-proteome (e.g., the phosphoproteome) levels. MS-based proteomics can reveal changes in the quantitative state of a proteome and provide a foundation for understanding the mechanisms involved in plant–pathogen interactions. This review is intended as a primer for biologists that may be unfamiliar with the diverse range of methodology for MS-based shotgun proteomics, with a focus on techniques that have been used to investigate plant–pathogen interactions. We provide a summary of the essential steps required for shotgun proteomic studies of plants, pathogens and plant–pathogen interactions, including methods for protein digestion, identification, separation, and quantification. Finally, we discuss how protein PTMs may directly participate in the interaction between a pathogen and its host plant.
]]>Proteomes doi: 10.3390/proteomes10010004
Authors: Arantxa Acera Juan Carlos Gómez-Esteban Ane Murueta-Goyena Marta Galdos Mikel Azkargorta Felix Elortza Noelia Ruzafa Oliver Ibarrondo Xandra Pereiro Elena Vecino
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n = 27) subjects was analyzed to identify candidate biomarkers for the diagnosis of PD. An observational, prospective and case-control pilot study was carried out, analyzing the participants tear samples by nano-liquid chromatography–mass spectrometry (nLC–MS/MS) and assessing their neurological impairment. The proteomic data obtained are available at ProteomeXchange with identifier 10.6019/PXD028811. These analyses led to the identification of 560 tear proteins, some of which were deregulated in PD patients and that have been implicated in immune responses, inflammation, apoptosis, collagen degradation, protein synthesis, defense, lipid transport and altered lysosomal function. Of these proteins, six were related to neurodegenerative processes and showed a good capacity to classify patients and controls. These findings revealed that certain proteins were upregulated in the tears of PD patients, mainly proteins involved in lysosomal function. Thus, in this study, tear proteins were identified that are implicated in neurodegeneration and that may be related to an aggressive disease phenotype in PD patients.
]]>Proteomes doi: 10.3390/proteomes10010003
Authors: Benjamin C. Orsburn Sierra D. Miller Conor J. Jenkins
Multiplexed proteomics using isobaric tagging allows for simultaneously comparing the proteomes of multiple samples. In this technique, digested peptides from each sample are labeled with a chemical tag prior to pooling sample for LC-MS/MS with nanoflow chromatography (NanoLC). The isobaric nature of the tag prevents deconvolution of samples until fragmentation liberates the isotopically labeled reporter ions. To ensure efficient peptide labeling, large concentrations of labeling reagents are included in the reagent kits to allow scientists to use high ratios of chemical label per peptide. The increasing speed and sensitivity of mass spectrometers has reduced the peptide concentration required for analysis, leading to most of the label or labeled sample to be discarded. In conjunction, improvements in the speed of sample loading, reliable pump pressure, and stable gradient construction of analytical flow HPLCs has continued to improve the sample delivery process to the mass spectrometer. In this study we describe a method for performing multiplexed proteomics without the use of NanoLC by using offline fractionation of labeled peptides followed by rapid “standard flow” HPLC gradient LC-MS/MS. Standard Flow Multiplexed Proteomics (SFloMPro) enables high coverage quantitative proteomics of up to 16 mammalian samples in about 24 h. In this study, we compare NanoLC and SFloMPro analysis of fractionated samples. Our results demonstrate that comparable data is obtained by injecting 20 µg of labeled peptides per fraction with SFloMPro, compared to 1 µg per fraction with NanoLC. We conclude that, for experiments where protein concentration is not strictly limited, SFloMPro is a competitive approach to traditional NanoLC workflows with improved up-time, reliability and at a lower relative cost per sample.
]]>Proteomes doi: 10.3390/proteomes10010002
Authors: Aarón Millán-Oropeza Mélisande Blein-Nicolas Véronique Monnet Michel Zivy Céline Henry
In proteomics, it is essential to quantify proteins in absolute terms if we wish to compare results among studies and integrate high-throughput biological data into genome-scale metabolic models. While labeling target peptides with stable isotopes allow protein abundance to be accurately quantified, the utility of this technique is constrained by the low number of quantifiable proteins that it yields. Recently, label-free shotgun proteomics has become the “gold standard” for carrying out global assessments of biological samples containing thousands of proteins. However, this tool must be further improved if we wish to accurately quantify absolute levels of proteins. Here, we used different label-free quantification techniques to estimate absolute protein abundance in the model yeast Saccharomyces cerevisiae. More specifically, we evaluated the performance of seven different quantification methods, based either on spectral counting (SC) or extracted-ion chromatogram (XIC), which were applied to samples from five different proteome backgrounds. We also compared the accuracy and reproducibility of two strategies for transforming relative abundance into absolute abundance: a UPS2-based strategy and the total protein approach (TPA). This study mentions technical challenges related to UPS2 use and proposes ways of addressing them, including utilizing a smaller, more highly optimized amount of UPS2. Overall, three SC-based methods (PAI, SAF, and NSAF) yielded the best results because they struck a good balance between experimental performance and protein quantification.
]]>Proteomes doi: 10.3390/proteomes10010001
Authors: Kira Vyatkina
De novo sequencing is indispensable for the analysis of proteins from organisms with unknown genomes, novel splice variants, and antibodies. However, despite a variety of methods developed to this end, distinguishing between the correct interpretation of a mass spectrum and a number of incorrect alternatives often remains a challenge. Tag convolution is computed for a set of peptide sequence tags of a fixed length k generated from the input tandem mass spectra and can be viewed as a generalization of the well-known spectral convolution. We demonstrate its utility for validating de novo peptide sequences by using a set of those generated by the algorithm PepNovo+ from high-resolution bottom-up data sets for carbonic anhydrase 2 and the Fab region of alemtuzumab and indicate its further potential applications.
]]>Proteomes doi: 10.3390/proteomes9040049
Authors: Ralph Wendt Justyna Siwy Tianlin He Agnieszka Latosinska Thorsten Wiech Peter F. Zipfel Aggeliki Tserga Antonia Vlahou Harald Rupprecht Lorenzo Catanese Harald Mischak Joachim Beige
Defective complement activation has been associated with various types of kidney disease. This led to the hypothesis that specific urine complement fragments may be associated with kidney disease etiologies, and disease progression may be reflected by changes in these complement fragments. We investigated the occurrence of complement fragments in urine, their association with kidney function and disease etiology in 16,027 subjects, using mass spectrometry based peptidomics data from the Human Urinary Proteome/Peptidome Database. Twenty-three different urinary peptides originating from complement proteins C3, C4 and factor B (CFB) could be identified. Most C3-derived peptides showed inverse association with estimated glomerular filtration rate (eGFR), while the majority of peptides derived from CFB demonstrated positive association with eGFR. Several peptides derived from the complement proteins C3, C4 and CFB were found significantly associated with specific kidney disease etiologies. These peptides may depict disease-specific complement activation and could serve as non-invasive biomarkers to support development of complement interventions through assessing complement activity for patients’ stratification and monitoring of drug impact. Further investigation of these complement peptides may provide additional insight into disease pathophysiology and could possibly guide therapeutic decisions, especially when targeting complement factors.
]]>Proteomes doi: 10.3390/proteomes9040048
Authors: Tossaporn Incharoen Sittiruk Roytrakul Wirot Likittrakulwong
Germinated paddy rice (GPR) could be a good alternative feed source for poultry with stocking density and heat stress problems. A total of 72 Hy-line Brown laying hens raised under low (LSD, 0.12 m2/bird) and high stocking densities (HSD, 0.06 m2/bird) were investigated. Three dietary GPR levels (0, 74 and 148 g/kg) were used. It was found that average daily feed intake, hen-day egg production, and egg mass significantly decreased in the HSD group. The levels of serum glucose (GLU), phosphorous (P), corticosterone (CORT), total Ig, lysozyme (LZY), and superoxide dismutase activities (SOD) in the HSD group were higher than those in the LSD group. Dietary GPR significantly affected GLU, P, alternative complement haemolytic 50 (ACH50), total Ig, and LZY. Moreover, CORT level significantly decreased in 74 and 148 g/kg dietary GPR groups, whereas SOD significantly increased only in the 148 g/kg dietary GPR group. Serum samples were analyzed using liquid chromatography-tandem mass spectrometry, and 8607 proteins were identified. Proteome analysis revealed 19 proteins which were enriched in different stocking densities and dietary GPR levels. Quantitative real-time reverse transcription-PCR technique was successfully used to verify the differentiated abundant protein profile changes. The proteins identified in this study could serve as appropriate biomarkers.
]]>Proteomes doi: 10.3390/proteomes9040047
Authors: Lou-Ann C. Andersen Nicolai Bjødstrup Palstrøm Axel Diederichsen Jes Sanddal Lindholt Lars Melholt Rasmussen Hans Christian Beck
Specific plasma proteins serve as valuable markers for various diseases and are in many cases routinely measured in clinical laboratories by fully automated systems. For safe diagnostics and monitoring using these markers, it is important to ensure an analytical quality in line with clinical needs. For this purpose, information on the analytical and the biological variation of the measured plasma protein, also in the context of the discovery and validation of novel, disease protein biomarkers, is important, particularly in relation to for sample size calculations in clinical studies. Nevertheless, information on the biological variation of the majority of medium-to-high abundant plasma proteins is largely absent. In this study, we hypothesized that it is possible to generate data on inter-individual biological variation in combination with analytical variation of several hundred abundant plasma proteins, by applying LC-MS/MS in combination with relative quantification using isobaric tagging (10-plex TMT-labeling) to plasma samples. Using this analytical proteomic approach, we analyzed 42 plasma samples prepared in doublets, and estimated the technical, inter-individual biological, and total variation of 265 of the most abundant proteins present in human plasma thereby creating the prerequisites for power analysis and sample size determination in future clinical proteomics studies. Our results demonstrated that only five samples per group may provide sufficient statistical power for most of the analyzed proteins if relative changes in abundances >1.5-fold are expected. Seventeen of the measured proteins are present in the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Biological Variation Database, and demonstrated remarkably similar biological CV’s to the corresponding CV’s listed in the EFLM database suggesting that the generated proteomic determined variation knowledge is useful for large-scale determination of plasma protein variations.
]]>Proteomes doi: 10.3390/proteomes9040046
Authors: Mário Jorge Araújo Maria Lígia Sousa Aldo Barreiro Felpeto Maria V. Turkina Elza Fonseca José Carlos Martins Vítor Vasconcelos Alexandre Campos
Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC–MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies; however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).
]]>Proteomes doi: 10.3390/proteomes9040045
Authors: Jennifer J. Hill Arsalan S. Haqqani Danica B. Stanimirovic
Interrogation of the molecular makeup of the blood–brain barrier (BBB) using proteomic techniques has contributed to the cataloguing and functional understanding of the proteins uniquely organized at this specialized interface. The majority of proteomic studies have focused on cellular components of the BBB, including cultured brain endothelial cells (BEC). Detailed proteome mapping of polarized BEC membranes and their intracellular endosomal compartments has led to an improved understanding of the processes leading to internalization and transport of various classes of molecules across the BBB. Quantitative proteomic methods have further enabled absolute and comparative quantification of key BBB transporters and receptors in isolated BEC and microvessels from various species. However, translational studies further require in vivo/in situ analyses of the proteins exposed on the luminal surface of BEC in vessels under various disease and treatment conditions. In vivo proteomics approaches, both profiling and quantitative, usually rely on ‘capturing’ luminally-exposed proteins after perfusion with chemical labeling reagents, followed by analysis with various mass spectrometry-based approaches. This manuscript reviews recent advances in proteomic analyses of luminal membranes of BEC in vitro and in vivo and their applications in translational studies focused on developing novel delivery methods across the BBB.
]]>Proteomes doi: 10.3390/proteomes9040044
Authors: Venus Baghalabadi Habib Razmi Alan Doucette
Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.
]]>Proteomes doi: 10.3390/proteomes9040043
Authors: Mariya E. Semkova J. Justin Hsuan
Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.
]]>Proteomes doi: 10.3390/proteomes9040042
Authors: Paul Dowling Ciara Tierney Katie Dunphy Juho J. Miettinen Caroline A. Heckman Despina Bazou Peter O’Gorman
Acute myeloid leukemia (AML) is characterized by an increasing number of clonal myeloid blast cells which are incapable of differentiating into mature leukocytes. AML risk stratification is based on genetic background, which also serves as a means to identify the optimal treatment of individual patients. However, constant refinements are needed, and the inclusion of significant measurements, based on the various omics approaches that are currently available to researchers/clinicians, have the potential to increase overall accuracy with respect to patient management. Using both nontargeted (label-free mass spectrometry) and targeted (multiplex immunoassays) proteomics, a range of proteins were found to be significantly changed in AML patients with different genetic backgrounds. The inclusion of validated proteomic biomarker panels could be an important factor in the prognostic classification of AML patients. The ability to measure both cellular and secreted analytes, at diagnosis and during the course of treatment, has advantages in identifying transforming biological mechanisms in patients, assisting important clinical management decisions.
]]>Proteomes doi: 10.3390/proteomes9040041
Authors: Elelwani Ramulifho Marie Emma Christine Rey
The production of cassava is threatened by the geminivirus South African cassava mosaic virus (SACMV), which causes cassava mosaic disease. Cassava landrace TME3 shows tolerance to SACMV, while T200 is highly susceptible. This study aimed to identify the leaf proteome involved in anti-viral defence. Liquid chromatography mass spectrometry (LC-MS) identified 2682 (54 differentially expressed) and 2817 (206 differentially expressed) proteins in both landraces at systemic infection (32 days post infection) and symptom recovery (67 days post infection), respectively. Differences in the number of differentially expressed proteins (DEPs) between the two landraces were observed. Gene ontology analysis showed that defence-associated pathways such as the chloroplast, proteasome, and ribosome were overrepresented at 67 days post infection (dpi) in SACMV-tolerant TME3. At 67 dpi, a high percentage (56%) of over-expressed proteins were localized in the chloroplast in TME3 compared to T200 (31% under-expressed), proposing that chloroplast proteins play a role in tolerance in TME3. Ribosomal_L7Ae domain-containing protein (Manes.12G139100) was over-expressed uniquely in TME3 at 67 dpi and interacts with the ribosomal protein Sac52 (RPL10). RPL10 is a known key player in the NIK1-mediated effector triggered immunity (ETI) response to geminivirus infection, indicating a possible role for Sac52 in SACMV recovery in TME3. In conclusion, differential protein expression responses in TME3 and T200 may be key to unravel tolerance to CMD.
]]>Proteomes doi: 10.3390/proteomes9040040
Authors: Wai Shun Mak Tsz Ming Tsang Tsz Yin Chan Georgi L. Lukov
This study investigates whether selected WD40 proteins with a 7-bladed β-propeller structure, similar to that of the β subunit of the G protein heterotrimer, interact with the cytosolic chaperonin CCT and its known binding partner, PhLP1. Previous studies have shown that CCT is required for the folding of the Gβ subunit and other WD40 proteins. The role of PhLP1 in the folding of Gβ has also been established, but it is unknown if PhLP1 assists in the folding of other Gβ-like proteins. The binding of three Gβ-like proteins, TBL2, MLST8 and CDC20, to CCT and PhLP1, was demonstrated in this study. Co-immunoprecipitation assays identified one novel binding partner for CCT and three new interactors for PhLP1. All three of the studied proteins interact with CCT and PhLP1, suggesting that these proteins may have a folding machinery in common with that of Gβ and that the well-established Gβ folding mechanism may have significantly broader biological implications than previously thought. These findings contribute to continuous efforts to determine common traits and unique differences in the folding mechanism of the WD40 β-propeller protein family, and the role PhLP1 has in this process.
]]>Proteomes doi: 10.3390/proteomes9040039
Authors: Yusuke Murashita Takumi Nishiuchi Shafiq Ur Rehman Setsuko Komatsu
Plant-derived smoke solution enhances soybean root growth; however, its mechanism is not clearly understood. Subcellular proteomics techniques were used for underlying roles of plant-derived smoke solution on soybean root growth. The fractions of membrane and nucleus were purified and evaluated for purity. ATPase and histone were enriched in the fractions of membrane and nucleus, respectively. Principal component analysis of proteomic results indicated that the plant-derived smoke solution affected the proteins in the membrane and nucleus. The proteins in the membrane and nucleus mainly increased and decreased, respectively, by the treatment of plant-derived smoke solution compared with control. In the proteins in the plasma membrane, ATPase increased, which was confirmed by immunoblot analysis, and ATP contents increased through the treatment of plant-derived smoke solution. Additionally, although the nuclear proteins mainly decreased, the expression of RNA polymerase II was up-regulated through the treatment of plant-derived smoke solution. These results indicate that plant-derived smoke solution enhanced soybean root growth through the transcriptional promotion with RNA polymerase II expression and the energy production with ATPase accumulation.
]]>Proteomes doi: 10.3390/proteomes9030038
Authors: Katrina Carbonara Martin Andonovski Jens R. Coorssen
Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must assess the myriad of proteoforms that arise at any point before, after, and between translation and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as newly defined species. There are numerous analytical methods currently used to address proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. We must embrace the complexity. Yes, these are the hard questions, and this will not be easy…but where is the fun in easy?
]]>Proteomes doi: 10.3390/proteomes9030037
Authors: Alba Gonzalez-Franquesa Lone Peijs Daniel Cervone Ceren Koçana Juleen Zierath Atul Deshmukh
Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR—an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.
]]>Proteomes doi: 10.3390/proteomes9030036
Authors: Betty Tijms Johan Gobom Charlotte Teunissen Valerija Dobricic Magda Tsolaki Frans Verhey Julius Popp Pablo Martinez-Lage Rik Vandenberghe Alberto Lleó José Molinuévo Sebastiaan Engelborghs Yvonne Freund-Levi Lutz Froelich Lars Bertram Simon Lovestone Johannes Streffer Stephanie Vos ADNI Kaj Blennow Philip Scheltens Henrik Zetterberg Pieter Visser
We recently discovered three distinct pathophysiological subtypes in Alzheimer’s disease (AD) using cerebrospinal fluid (CSF) proteomics: one with neuronal hyperplasticity, a second with innate immune system activation, and a third subtype with blood–brain barrier dysfunction. It remains unclear whether AD proteomic subtype profiles are a consequence of amyloid aggregation, or might exist upstream from aggregated amyloid. We studied this question in 127 older individuals with intact cognition and normal AD biomarkers in two independent cohorts (EMIF-AD MBD and ADNI). We clustered 705 proteins measured in CSF that were previously related to AD. We identified in these cognitively intact individuals without AD pathology three subtypes: two subtypes were seen in both cohorts (n = 49 with neuronal hyperplasticity and n = 44 with blood–brain barrier dysfunction), and one only in ADNI (n = 12 with innate immune activation). The proteins specific for these subtypes strongly overlapped with AD subtype protein profiles (overlap coefficients 92%–71%). Longitudinal p181-tau and amyloid β 1–42 (Aβ42) CSF analysis showed that in the hyperplasticity subtype p181-tau increased (β = 2.6 pg/mL per year, p = 0.01) and Aβ42 decreased over time (β = −4.4 pg/mL per year, p = 0.03), in the innate immune activation subtype p181-tau increased (β = 3.1 pg/mL per year, p = 0.01) while in the blood–brain barrier dysfunction subtype Aβ42 decreased (β = −3.7 pg/mL per year, p = 0.009). These findings suggest that AD proteomic subtypes might already manifest in cognitively normal individuals and may predispose for AD before amyloid has reached abnormal levels.
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