Search Results (92)

Botrytis cinerea is a highly versatile pathogenic fungus, causing significant damage across a wide range of plant species. A central focus of this review is the recent advances made through proteomics, an advanced molecular tool, in understanding the mechanisms of B. cinerea infection. Recent advances in mass spectrometry-based proteomics—including LC-MS/MS, iTRAQ, MALDI-TOF, and surface shaving—have enabled the in-depth characterization of B. cinerea subproteomes such as the secretome, surfactome, phosphoproteome, and extracellular vesicles, revealing condition-specific pathogenic mechanisms. Notably, in under a decade, the proportion of predicted proteins experimentally identified has increased from 10% to 52%, reflecting the rapid progress in proteomic capabilities. We explore how proteomic studies have significantly enhanced our knowledge of the fungus secretome and the role of extracellular vesicles (EVs), which play key roles in pathogenesis, by identifying secreted proteins—such as pH-responsive elements—that may serve as biomarkers and therapeutic targets. These technologies have also uncovered fine regulatory mechanisms across multiple levels of the fungal proteome, including post-translational modifications (PTMs), the phosphomembranome, and the surfactome, providing a more integrated view of its infection strategy. Moreover, proteomic approaches have contributed to a better understanding of host–pathogen interactions, including aspects of the plant’s defensive responses. Furthermore, this review discusses how proteomic data have helped to identify metabolic pathways affected by novel, more environmentally friendly antifungal compounds. A further update on the advances achieved in the field of proteomics discovery for the organism under consideration is provided in this paper, along with a perspective on emerging tools and future developments expected to accelerate research and improve targeted intervention strategies. Full article

Over the past decade, the number and diversity of identified protein post-translational modifications (PTMs) have grown significantly. However, most PTMs occur at relatively low abundance, making selective enrichment of modified peptides essential. To address this, we developed a thermodynamic model describing the free beads enrichment in suspension enrichment process and derived a theoretical relationship between material dosage and analyte recovery. The model predicts a non-linear trend, with enrichment efficiency increasing up to an optimal dosage and declining thereafter—a pattern confirmed by experimental data. We validated the model using centrifugation-based enrichment for glycosylated peptides and magnetic-based enrichment for phosphorylated peptides. In both cases, the results aligned with theoretical predictions. Additionally, the optimal dosage varied among peptides with the same modification type, highlighting the importance of tailoring enrichment strategies. This study provides a solid theoretical and experimental basis for optimizing PTMs enrichment and advancing more sensitive, accurate, and efficient mass spectrometry-based proteomic workflows. Full article

Redox signaling is central to plant adaptation, influencing metabolic regulation, stress responses, and developmental processes through thiol-based oxidative post-translational modifications (oxiPTMs) of redox-sensitive proteins. These modifications, particularly those involving cysteine (Cys) residues, act as molecular switches that alter protein function, structure, and interactions. Advances in mass spectrometry-based redox proteomics have greatly enhanced the identification and quantification of oxiPTMs, enabling a more refined understanding of redox dynamics in plant cells. In parallel, the emergence of computational modeling, artificial intelligence (AI), and machine learning (ML) has revolutionized the ability to predict redox-sensitive residues and characterize redox-dependent signaling networks. This review provides a comprehensive synthesis of methodological advancements in redox proteomics, including enrichment strategies, quantification techniques, and real-time redox sensing technologies. It also explores the integration of computational tools for predicting S-nitrosation, sulfenylation, S-glutathionylation, persulfidation, and disulfide bond formation, highlighting key models such as CysQuant, BiGRUD-SA, DLF-Sul, and Plant PTM Viewer. Furthermore, the functional significance of redox modifications is examined in plant development, seed germination, fruit ripening, and pathogen responses. By bridging experimental proteomics with AI-driven prediction platforms, this review underscores the future potential of integrated redox systems biology and emphasizes the importance of validating computational predictions, through experimental proteomics, for enhancing crop resilience, metabolic efficiency, and precision agriculture under climate variability. Full article

Peroxiredoxins (Prxs; EC 1.11.1.15) are a group of thiol peroxidases that catalyze the detoxification of H₂O₂ and other organic hydroperoxides. The ripening of pepper (Capsicum annuum L.) fruit involves significant phenotypic, physiological, and biochemical changes. Based on the available pepper plant genome, eight PRX genes were identified and named CaPRX1, CaPRX1-Cys, CaPRX2B, CaPRX2E, CaPRX2F, CaPRX2-CysBAS1, CaPRX2-CysBAS2, and CaPRX Q. Among these, only CaPRX1-Cys was not detected in the transcriptome (RNA-Seq) of sweet pepper fruits reported previously. This study analyzes the modulation of these seven CaPRX genes during ripening and after treating fruits with nitric oxide (NO) gas. A time-course expression analysis of sweet pepper fruit during ripening revealed that two genes were upregulated (CaPRX1 and CaPRX2E), two were downregulated (CaPRX2B and PRX Q), and three were unaffected (CaPRX2F, CaPRX2-CysBAS1, and CaPRX2-CysBAS2). Gene expression was also studied in three hot pepper varieties with varying capsaicin contents (Piquillo < Padrón < Alegría riojana), showing a differential expression pattern during ripening. Furthermore, NO treatment of sweet pepper fruits triggered the upregulation of CaPRX2B and CaPRXQ genes and the downregulation of CaPRX1 and CaPRX2-CysBAS1 genes, while the other three remained unaffected. Among the CaPrx proteins, four (CaPrx2B, CaPrx2-CysBAS1, CaPrx2-CysBAS2, and CaPrx2E) were identified as susceptible to S-nitrosation, as determined by immunoprecipitation assays with an antibody against S-nitrocysteine and further mass spectrometry analyses. These findings indicate the diversification of PRX genes in pepper fruits and how some of them are regulated by NO, either at the level of gene expression or through protein S-nitrosation, a NO-promoting post-translational modification (PTM). Given that Prxs play a crucial role in stress tolerance, these data suggest that Prxs are vital components of the antioxidant system during pepper fruit ripening, an event that is accompanied by physiological nitro-oxidative stress. Full article

Background/Objectives:Cancer vaccine targets mostly include mutations and overexpressed proteins. However, cancer-associated post-translational modifications (PTMs) may also induce immune responses. Previously, our group established the enzyme protein arginine deiminase type-2 (PADI2), which catalyzes citrullination modification, is highly expressed in triple-negative breast cancer (TNBC), promoting antigenicity. Methods: Here, we show the workflow of designing citrullinated enolase 1 (citENO1) vaccine peptides identified from breast cancer cells by mass spectrometry and demonstrate TNBC vaccine efficacy in the mouse model. Immunized mice with citENO1 peptides or the corresponding unmodified peptides, plus Poly I:C as an adjuvant, were orthotopically implanted with a TNBC murine cell line. Results: Vaccination with citENO1, but not unmodified ENO1 (umENO1), induced a greater percentage of activated CD8+ PD-1+ T cells and effector memory T cells in skin-draining lymph nodes (SDLNs). Remarkably, the citENO1 vaccine delayed tumor growth and prolonged overall survival, which was further enhanced by PD-1 blockade. Conclusions: Our data suggest that cancer-restricted post-translational modifications provide a source of vaccines that induce an anti-cancer immune response. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, heterogeneous at the molecular level and characterized by diverse and complex pathological features. Such features are known to accumulate silently in the brain over years or even decades before the onset of detectable symptoms. Despite long years of intense research activities, the disease remains orphaned of either disease-modifying therapies or a specific blood test capable of predicting the disease in the pre-symptomatic stages. This disappointing outcome of such efforts can be attributed to a number of factors. One of these factors is the failure of earlier research to capture the heterogeneity of the disease. Such failure has the direct consequence of poor patient stratification, which in turn impacts negatively on the development of specific and effective therapy. The second factor is the absence of detailed and accurate information on proteins and associated post-translational modifications, which may influence the initiation and progress of the disease. Recent studies have demonstrated that the quantification of various isoforms of phosphorylated tau protein in plasma and other biofluids can be considered as potential biomarkers for the early detection of Alzheimer’s disease. Mass spectrometry-based proteomics and immunoassay-based multiplex proteomics are the two technologies in current use for probing the human proteome, both in tissues and biofluids. In the present review, we discuss the contribution of MS-based proteomics to efforts aimed at the identification and eventual characterization of the heterogeneity of the disease, and the key role of the same technique in the analysis of protein post-translational modifications associated with the disease is also discussed. Full article

Background/Objectives: Multi-dimensional liquid chromatography coupled with mass spectrometry (mD-LC-MS) has emerged as a powerful technique for the in-depth characterization of biopharmaceuticals by assessing chromatographically resolved product variants in a streamlined and semi-automated manner. The study aims to demystify and enhance the accessibility to this powerful but inherently complex technique by detailing a robust and user-friendly instrument platform, allowing analysts to switch seamlessly between intact, subunit, and peptide mapping workflows. Methods: Starting from a commercially available Two-Dimensional Liquid Chromatography (2D-LC) system, we introduce specific hardware and software extensions leading to two versatile mD-LC-MS setups, in slightly different configurations. The technique’s efficacy is demonstrated through a case study on a cation exchange chromatography method assessing the charge variants of a bispecific antibody, isolating peak(s) of interest, followed by online sample processing, including reduction and enzymatic digestion, and subsequently mass spectrometry analysis. Results: The accuracy and reproducibility of both mD-LC-MS setups proposed in this study were successfully tested. Despite the complex peak patterns in the first dimension, the systems were equally effective in identifying and quantifying the underlying product species. This case study highlights the routine usability of mD-LC-MS technology for the characterization of (ultra) high-performance liquid chromatography (UHPLC) of therapeutic biomolecule. Conclusions: The demonstrated reliability and accuracy underscore the practicality of mD-LC-MS for routine use in biopharmaceutical analysis. Our detailed description of the mD-LC-MS systems and insights simplify access to this advanced technology for a broader scientific community, regardless of expertise level, and lower the entry barrier for its use in various research and industrial settings. Full article

Nelumbo nucifera is an aquatic plant with a high ornamental value due to its flower. Despite the release of several versions of the lotus genome, its annotation remains inefficient, which makes it difficult to obtain a more comprehensive knowledge when –omic studies are applied to understand the different biological processes. Focusing on the petaloid of the lotus flower, we conducted a comparative proteomic analysis among five major floral organs. The proteogenomic strategy was applied to analyze the mass spectrometry data in order to dig out novel proteoforms that are involved in the petaloids of the lotus flower. The results revealed that a total of 4863 proteins corresponding to novel genes were identified, with 227 containing single amino acid variants (SAAVs), and 72 originating from alternative splicing (AS) genes. In addition, a range of post-translational modifications (PTMs) events were also identified in lotus. Through functional annotation and homology analysis with 24 closely related plant species, we identified five candidate proteins associated with floral organ development, which were not identified by ordinary proteomic analysis. This study not only provides new insights into understanding the mechanism of petaloids in lotus but is also helpful in identifying new proteoforms to improve the annotation of the lotus genome. Full article

Corynebacterium glutamicum (C. glutamicum) has become industrially important in producing glutamic acid and lysine since its discovery and has been the subject of proteomics and central carbon metabolism studies. The proteome changes depending on environmental conditions, nutrient availability, and stressors. Post-translational modification (PTMs), such as phosphorylation, methylation, and glycosylation, alter the function and activity of proteins, allowing them to respond quickly to environmental changes. Proteomics techniques, such as mass spectrometry and two-dimensional gel electrophoresis, have enabled the study of proteomes, identification of proteins, and quantification of the expression levels. Understanding proteomes and central carbon metabolism in microorganisms provides insight into their physiology, ecology, and biotechnological applications, such as biofuels, pharmaceuticals, and industrial enzyme production. Several attempts have been made to create efficient production strains to increase productivity in several research fields, such as genomics and proteomics. In addition to amino acids, C. glutamicum is used to produce vitamins, nucleotides, organic acids, and alcohols, expanding its industrial applications. Considerable information has been accumulated, but recent research has focused on proteomes and central carbon metabolism. The development of genetic engineering technologies, such as CRISPR-Cas9, has improved production efficiency by allowing precise manipulation of the metabolic pathways of C. glutamicum. In addition, methods for designing new metabolic pathways and developing customized strains using synthetic biology technology are gradually expanding. This review is expected to enhance the understanding of C. glutamicum and its industrial potential and help researchers identify research topics and design studies. Full article

Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies in one solution. It is extremely difficult to effectively separate the charge variants of the two co-formulated antibodies using one ion exchange chromatography (IEC) method because of their similar characteristics. In this study, a novel method was developed for the charge variants characterization of co-formulated antibodies using three-dimensional liquid chromatography–mass spectrometry (3D-LC-MS). Hydrophobic interaction chromatography (HIC) was used as the first dimension to separate and collect the two co-formulated antibodies. The two collections were then injected into the second-dimension IEC separately for charge variants separation and analysis. Subsequently, the separated charge variants underwent on-line desalting in the third-dimension reverse-phase chromatography (RPC) and subsequent mass spectroscopy analysis. The novel method could simultaneously provide a charge variants ratio and post-translational modification (PTM) data for the two co-formulated antibodies. Therefore, it could be used for release testing and stability studies of co-formulated antibodies, making up for the shortcomings of the existing approaches. It was the first time that charge variants of co-formulated antibodies were characterized by the 3D-LC-MS method, to the best of our knowledge. Full article

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17β-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography–tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC. Full article

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift. Full article

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H₂S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H₂S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits. Full article

The high-throughput proteomics data generated by increasingly more sensible mass spectrometers greatly contribute to our better understanding of molecular and cellular mechanisms operating in live beings. Nevertheless, proteomics analyses are based on accurate genomic and protein annotations, and some information may be lost if these resources are incomplete. Here, we show that most proteomics data may be recovered by interconnecting genomics and proteomics approaches (i.e., following a proteogenomic strategy), resulting, in turn, in an improvement of gene/protein models. In this study, we generated proteomics data from Leishmania donovani (HU3 strain) promastigotes that allowed us to detect 1908 proteins in this developmental stage on the basis of the currently annotated proteins available in public databases. However, when the proteomics data were searched against all possible open reading frames existing in the L. donovani genome, twenty new protein-coding genes could be annotated. Additionally, 43 previously annotated proteins were extended at their N-terminal ends to accommodate peptides detected in the proteomics data. Also, different post-translational modifications (phosphorylation, acetylation, methylation, among others) were found to occur in a large number of Leishmania proteins. Finally, a detailed comparative analysis of the L. donovani and Leishmania major experimental proteomes served to illustrate how inaccurate conclusions can be raised if proteomes are compared solely on the basis of the listed proteins identified in each proteome. Finally, we have created data entries (based on freely available repositories) to provide and maintain updated gene/protein models. Raw data are available via ProteomeXchange with the identifier PXD051920. Full article

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography–tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development. Full article