Proteomes, Volume 11, Issue 4 (December 2023) – 12 articles

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article