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SDS Depletion from Intact Membrane Proteins by KCl Precipitation Ahead of Mass Spectrometry Analysis
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Novel Serum Glycoprotein Biomarkers Clinically Validated as a Diagnostic Test for Esophageal Adenocarcinoma
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Deciphering Radiotherapy Resistance: A Proteomic Perspective
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The Proteomic Signature of Oxidative Stress in Myelodysplastic Syndromes
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Integrative Spatial Proteomics and Single-Cell RNA Sequencing Unveil Molecular Complexity in Rheumatoid Arthritis for Novel Therapeutic Targeting
Journal Description
Proteomes
Proteomes
is an international, peer-reviewed, open access journal on all aspects of proteomics published quarterly online by MDPI.
- Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
- High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, CAPlus / SciFinder, and other databases.
- Journal Rank: JCR - Q2 (Biochemistry and Molecular Biology) / CiteScore - Q1 (Structural Biology)
- Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 28.2 days after submission; acceptance to publication is undertaken in 4.8 days (median values for papers published in this journal in the first half of 2025).
- Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.
Impact Factor:
3.6 (2024);
5-Year Impact Factor:
3.9 (2024)
Latest Articles
Neurogranin as a Synaptic Biomarker in Mild Traumatic Brain Injury: A Systematic Review of Diagnostic and Pathophysiological Evidence
Proteomes 2025, 13(3), 46; https://doi.org/10.3390/proteomes13030046 - 19 Sep 2025
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Neurogranin (NRGN), a synaptic protein essential for plasticity and memory function, is gaining recognition as a promising biomarker for mild traumatic brain injury (mTBI). This systematic review brings together findings from six studies that measured neurogranin levels in biofluids—including serum, cerebrospinal fluid (CSF),
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Neurogranin (NRGN), a synaptic protein essential for plasticity and memory function, is gaining recognition as a promising biomarker for mild traumatic brain injury (mTBI). This systematic review brings together findings from six studies that measured neurogranin levels in biofluids—including serum, cerebrospinal fluid (CSF), plasma, and exosomes—during both the acute and chronic phases following injury. In the acute phase of mTBI, elevated levels of neurogranin were consistently observed in serum samples, suggesting its potential as a diagnostic marker. These increases appear to reflect immediate synaptic disturbances caused by injury. In contrast, studies focusing on the chronic phase reported a decrease in exosomal neurogranin levels, pointing to ongoing synaptic dysfunction well after the initial trauma. This temporal shift in neurogranin expression highlights its dual utility—both as an early indicator of injury and as a longer-term marker of synaptic integrity. However, interpreting these findings is not straightforward. The studies varied considerably in terms of sample type, timing of measurements, and control for potential confounding factors such as physical activity. Such variability makes direct comparisons difficult and may influence the outcomes observed. Additionally, none of the studies included proteoform-specific analyses of neurogranin, an omission that limits our understanding of the molecular changes underlying mTBI-related synaptic alterations. Due to heterogeneity across study designs and outcome measures, a meta-analysis could not be performed. Instead, a narrative synthesis was conducted, revealing consistent patterns in neurogranin dynamics over time and underscoring the influence of biofluid selection on measured outcomes. Overall, the current evidence supports neurogranin’s potential as both a diagnostic and mechanistic biomarker for mTBI. Yet, to fully realize its clinical utility, future research must prioritize standardized protocols, the inclusion of proteoform profiling, and rigorous longitudinal validation studies.
Full article
Open AccessArticle
Comparative Analysis of Plasma Extracellular Vesicle Isolation Methods for Purity Assessment and Biomarker Discovery
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Alexandra T. Star, Melissa Hewitt, Amanpreet Badhwar, Wen Ding, Tammy-Lynn Tremblay, Jennifer J. Hill, William G. Willmore, Jagdeep K. Sandhu and Arsalan S. Haqqani
Proteomes 2025, 13(3), 45; https://doi.org/10.3390/proteomes13030045 - 18 Sep 2025
Abstract
Background: Extracellular vesicles (EVs) are an important source of blood biomarkers and are emerging as next-generation therapeutics. Demonstrating the purity of isolated EVs is essential for applications ranging from proteomics-based biomarker discovery to biomanufacturing. In this study, we systematically evaluated multiple EV isolation
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Background: Extracellular vesicles (EVs) are an important source of blood biomarkers and are emerging as next-generation therapeutics. Demonstrating the purity of isolated EVs is essential for applications ranging from proteomics-based biomarker discovery to biomanufacturing. In this study, we systematically evaluated multiple EV isolation methods for plasma and developed a scoring method to identify the approach best suited for proteomics. Methods: Commonly used enrichment techniques, including size-exclusion chromatography (SEC) and precipitation-based methods, were compared against the starting plasma in terms of particle yield and size, proteomic overlap, depletion of abundant plasma proteins, and enrichment of EV markers and unique proteins. To enable rigorous purity assessment, we established a targeted parallel reaction monitoring (PRM) mass spectrometry assay that quantified key EV markers and contaminant proteins across preparations. Results: Among the methods tested, SEC showed the greatest enrichment of EV markers and unique proteins, with the lowest level of contaminants, resulting in the highest overall purity scores. SEC also allowed for the detection of EV-free proteins. Other methods, by contrast, performed sub-optimally and were less reliable for proteomics-driven biomarker discovery. Conclusions: SEC provides the most EV-enriched plasma isolates for proteomics information, with minimal contamination from plasma proteins. The PRM-based purity scoring offers an objective means of benchmarking EV preparations and may help standardize EV isolation quality for both biomarker discovery and therapeutic manufacturing.
Full article
(This article belongs to the Topic Liquid Biopsy: A Modern Method Transforming Biomedicine)
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Open AccessArticle
Proteomic Analysis of Mechanical Injury Effects in Papaya Fruit at Two Maturity Stages
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Francisco Antonio Reyes-Soria, Eliel Ruiz-May, Enrique Castaño, Miguel Ángel Herrera-Alamillo, José Miguel Elizalde-Contreras, Samuel David Gamboa-Tuz, Lidia F. E. Huerta-Nuñez, Jesús Alejandro Zamora-Briseño and Luis Carlos Rodríguez-Zapata
Proteomes 2025, 13(3), 44; https://doi.org/10.3390/proteomes13030044 - 18 Sep 2025
Abstract
Background: Mechanical damage to fruit during harvesting is nearly inevitable, with certain species, such as papaya, being particularly prone to spoilage. Postharvest handling can induce mechanical injuries that impair ripening and reduce shelf life, leading to significant economic losses. Although several studies have
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Background: Mechanical damage to fruit during harvesting is nearly inevitable, with certain species, such as papaya, being particularly prone to spoilage. Postharvest handling can induce mechanical injuries that impair ripening and reduce shelf life, leading to significant economic losses. Although several studies have shed light on the molecular bases of mechanical damage, other aspects remain to be described (plant hormone inter-talk, physiological changes, and regulatory networks). Methods: In this study, we investigated proteomic changes in papaya fruit at two distinct ripening stages following mechanical damage. A total of 3230 proteins were identified, representing the most comprehensive proteomic analysis of papaya to date and the first assessment of proteins regulated by mechanical stress. Results: Proteins involved in ethylene biosynthesis were up-regulated on Day 2 but down-regulated on Day 12, with a similar trend observed for proteins in the abscisic acid synthesis pathway. Enzymes associated with photosynthesis, carbon fixation, primary metabolism, and carotenoid synthesis were down-regulated at both stages. In contrast, those related to plasmodesmata, calcium signaling, kinases, pathogenesis, cell wall remodeling, and proteases were up-regulated. Conclusions: These findings are thoroughly discussed, and a general model of the events triggered by mechanical impact in papaya is proposed. Our results provide a comprehensive framework for understanding papaya’s response to mechanical damage.
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(This article belongs to the Section Proteome Bioinformatics)
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Open AccessArticle
Proteomic Analysis of Sputum from Patients with Active Tuberculosis
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Endrei Marcantonio, Amy M. Woron, A. Christian Whelen and Sladjana Prisic
Proteomes 2025, 13(3), 43; https://doi.org/10.3390/proteomes13030043 - 12 Sep 2025
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Background: Patients with pulmonary tuberculosis (TB) typically produce sputa, which are used to identify the pathogen. Sputum also contains host proteins that may aid in diagnosis. We hypothesized that sputa from TB patients will have unique proteomes when compared to other lung diseases.
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Background: Patients with pulmonary tuberculosis (TB) typically produce sputa, which are used to identify the pathogen. Sputum also contains host proteins that may aid in diagnosis. We hypothesized that sputa from TB patients will have unique proteomes when compared to other lung diseases. Methods: Sputa were collected from 219 patients with suspected TB. Neutrophil-derived protein calprotectin (CP), which was used as a marker for lung damage, was quantified and compared between TB and non-TB groups. Three sputa with high or low CP from each group were selected and analyzed using label-free proteomics. Results: There was no difference in CP amounts between TB and non-TB groups. However, TB samples had other differentially abundant neutrophil-associated proteins. Compared to low CP, samples with high CP had much smaller number of proteins that could differentiate between TB and non-TB groups. Only two proteins, MUC5AC and MMP8, were more abundant in TB samples, regardless of CP levels. Conclusions: Our findings suggest that TB sputa may have unique proteomes that depend on CP levels, which should be further validated due to the small sample size. Therefore, controlled and more advanced TB may need a different set of biomarkers to reliably distinguish TB from other lung diseases.
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Open AccessArticle
Proteomic Analysis of the Periodontal Ligament During Orthodontic Movement: A Study in Rats
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Camila Chierici Marcantonio, Maria Eduarda Scordamaia Lopes, Lélio Fernando Ferreira Soares, Cristiane Ribeiro Salmon, Francisco Humberto Nociti Junior, James Deschner, Andressa Vilas Boas Nogueira and Joni Augusto Cirelli
Proteomes 2025, 13(3), 42; https://doi.org/10.3390/proteomes13030042 - 11 Sep 2025
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The periodontal ligament (PDL) is a dynamic connective tissue that absorbs and transmits mechanical forces, playing a critical role during orthodontic tooth movement (OTM). This study aimed to characterize the proteomic profile of rat PDLs subjected to OTM. Ten Holtzman rats were allocated
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The periodontal ligament (PDL) is a dynamic connective tissue that absorbs and transmits mechanical forces, playing a critical role during orthodontic tooth movement (OTM). This study aimed to characterize the proteomic profile of rat PDLs subjected to OTM. Ten Holtzman rats were allocated into Control and OTM groups. After 15 days of force application, hemimaxillae were harvested, and PDL tissues from the first maxillary molars were isolated via laser capture microdissection. Protein extracts were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), followed by quantitative and enrichment analyses. Immunohistochemistry was performed to validate selected proteins. The full proteomic datasets supporting these findings are available in the PRIDE repository under the identifiers PXD055817 and PXD033647. A total of 1121 proteins were identified; 101 were exclusive to the OTM group, 324 to the control, and 696 shared. Among the 335 proteins with differential abundance, 334 were downregulated and one (Prelp) was upregulated in the OTM group. Enrichment analysis revealed that differentially abundant proteins were associated with molecular functions such as protein binding, and cellular components including extracellular exosomes, focal adhesions, and the extracellular matrix. Immunohistochemical analysis confirmed the presence of Prelp, Rbm3, and Cirbp in PDL tissues. These findings demonstrate that OTM significantly alters the proteomic landscape of the PDL and identify key proteins potentially involved in periodontal remodeling.
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Open AccessArticle
Bidirectional Interaction Between PGE2-Preconditioned Mesenchymal Stem Cells and Myofibroblasts Mediates Anti-Fibrotic Effects: A Proteomic Investigation into Equine Endometrial Fibrosis Reversal
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Lidice Méndez-Pérez, Yat Sen Wong, Belén O. Ibáñez, Ioanna Martinez-Hormaza, Lleretny Rodríguez-Álvarez and Fidel Ovidio Castro
Proteomes 2025, 13(3), 41; https://doi.org/10.3390/proteomes13030041 - 8 Sep 2025
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Background: Endometrosis is a prevalent fibrotic condition in mares that impairs reproductive efficiency by inducing transdifferentiation of endometrial stromal cells into myofibroblasts, leading to excessive ECM deposition. Methods: To elucidate the molecular mechanisms underlying fibrosis resolution, this study employed comprehensive proteomic techniques, including
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Background: Endometrosis is a prevalent fibrotic condition in mares that impairs reproductive efficiency by inducing transdifferentiation of endometrial stromal cells into myofibroblasts, leading to excessive ECM deposition. Methods: To elucidate the molecular mechanisms underlying fibrosis resolution, this study employed comprehensive proteomic techniques, including LC-MS/MS and SILAC, to analyze the interaction between myofibroblasts and mesenchymal stem cells derived from the endometrium (ET-eMSCs) preconditioned with PGE2. An in vitro co-culture system was used, with samples collected at baseline and after 48 h. Results: Proteomic analysis identified significant alterations in proteins associated with ECM remodeling, immune regulation, and cellular stress response. Notably, proteins involved in collagen degradation, antioxidant defense, and growth factor signaling pathways were differentially abundant. Network analyses demonstrated robust interactions among these proteins, suggesting coordinated modulatory effects. The data indicate that PGE2-primed ET-eMSCs induce a shift in myofibroblast secretory profiles, promoting a reduction in ECM stiffness, tissue reorganization, and activation of resolution pathways. Data are available via ProteomeXchange with identifier PXD067551. Conclusions: These findings reinforce the therapeutic potential of mesenchymal stem cell-based interventions for fibrotic diseases of the endometrium, opening avenues for regenerative strategies to restore reproductive function in mares.
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Open AccessArticle
The Biological Variation in Serum ACE and CPN/CPB2 Activity in Healthy Individuals as Measured by the Degradation of Dabsylated Bradykinin—Reference Data and the Importance of Pre-Analytical Standardization
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Malte Bayer, Michael Snyder and Simone König
Proteomes 2025, 13(3), 40; https://doi.org/10.3390/proteomes13030040 - 27 Aug 2025
Abstract
Background: Bradykinin (BK) is an inflammatory mediator. The degradation of labeled synthetic BK in biofluids can be used to report on the activity of angiotensin-converting enzyme (ACE) and basic carboxypeptidases N and CBP2, for which the neuropeptide is a substrate. Clinical studies have
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Background: Bradykinin (BK) is an inflammatory mediator. The degradation of labeled synthetic BK in biofluids can be used to report on the activity of angiotensin-converting enzyme (ACE) and basic carboxypeptidases N and CBP2, for which the neuropeptide is a substrate. Clinical studies have shown significant changes in the serum activity of these enzymes in patients with inflammatory diseases. Methods: Here, we investigated variation in the cleavage of dabsylated synthetic BK (DBK) in serum and the formation of the major enzymatic fragments using a thin-layer chromatography-based neuropeptide reporter assay (NRA) in a large cohort of healthy volunteers from the international human Personal Omics Profiling consortium based at Stanford University. Results: Four major outcomes were reported. First, a set of NRA reference data for the healthy population was delivered, which is important for future investigations of patient sera. Second, it was shown that the measured serum degradation capacity for DBK was significantly higher in males than in females. There was no significant correlation of the NRA results with ethnicity, body mass index or overnight fasting. Third, a batch effect was noted among sampling sites (HUPO conferences). Thus, we used subcohorts rather than the entire collection for data mining. Fourth, as the low-cost and robust NRA is sensitive to enzyme activity, it provides such a necessary quick test to eliminate degraded and/or otherwise questionable samples. Conclusions: The results reiterate the critical importance of a high level of standardization in pre-analytical sample collection and processing—most notably, sample quality should be evaluated before conducting any large and expensive omics analyses.
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(This article belongs to the Section Proteomics Technology and Methodology Development)
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Adiposome Proteomics Uncover Molecular Signatures of Cardiometabolic Risk in Obese Individuals
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Mohamed Saad Rakab, Monica C. Asada, Imaduddin Mirza, Mohammed H. Morsy, Amro Mostafa, Francesco M. Bianco, Mohamed M. Ali, Chandra Hassan, Mario A. Masrur, Brian T. Layden and Abeer M. Mahmoud
Proteomes 2025, 13(3), 39; https://doi.org/10.3390/proteomes13030039 - 26 Aug 2025
Abstract
Background: Adipose-derived extracellular vesicles (adiposomes) are emerging as key mediators of inter-organ communication, yet their molecular composition and role in obesity-related pathophysiology remain underexplored. This study integrates clinical phenotyping with proteomic analysis of visceral adipose-derived adiposomes to identify obesity-linked molecular disruptions. Methods: Seventy-five
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Background: Adipose-derived extracellular vesicles (adiposomes) are emerging as key mediators of inter-organ communication, yet their molecular composition and role in obesity-related pathophysiology remain underexplored. This study integrates clinical phenotyping with proteomic analysis of visceral adipose-derived adiposomes to identify obesity-linked molecular disruptions. Methods: Seventy-five obese and forty-seven lean adults were extensively profiled for metabolic, inflammatory, hepatic, and vascular parameters. Adiposomes isolated from visceral fat underwent mass spectrometry-based proteomic analysis, followed by differential abundance, pathway enrichment, regulatory network modeling, and clinical association testing. Results: Obese individuals exhibited widespread cardiometabolic dysfunction. Proteomics revealed 64 adiposomal proteins with differential abundance. Upregulated proteins (e.g., CRP, C9, APOC1) correlated with visceral adiposity, systemic inflammation, and endothelial dysfunction. In contrast, downregulated proteins (e.g., ADIPOQ, APOD, TTR, FGB, FGG) were associated with enhanced nitric oxide bioavailability and vascular protection, suggesting loss of homeostatic signaling. Network analyses identified TNF and IL1 as key upstream regulators driving inflammatory and oxidative stress pathways. Decision tree and random forest models accurately classified obesity, hypertension, diabetes, dyslipidemia, and hepatic steatosis (AUC = 0.908–0.994), identifying predictive protein signatures related to complement activation, inflammation, and lipid transport. Conclusion: Obesity alters adiposome proteomic cargo, reflecting and potentially mediating systemic inflammation, metabolic dysregulation, and vascular impairment.
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(This article belongs to the Special Issue Proteomics in Chronic Diseases: Issues and Challenges)
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Open AccessArticle
Identification of Cancer-Associated Proteins in Colorectal Cancer Using Mass Spectrometry
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Naoyuki Toyota, Ryo Konno, Shuhei Iwata, Shin Fujita, Yoshio Kodera, Rei Noguchi, Tadashi Kondo, Yusuke Kawashima and Yuki Yoshimatsu
Proteomes 2025, 13(3), 38; https://doi.org/10.3390/proteomes13030038 - 12 Aug 2025
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Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with a multifactorial etiology involving genetic and environmental factors. Advanced proteomics offers valuable insights into the molecular mechanisms of cancer, identifying proteins that function as mediators in tumor biology. Methods: In
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Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with a multifactorial etiology involving genetic and environmental factors. Advanced proteomics offers valuable insights into the molecular mechanisms of cancer, identifying proteins that function as mediators in tumor biology. Methods: In this study, we used mass spectrometry-based data-independent acquisition (DIA) to analyze the proteomic landscape of CRC. We compared protein abundance in normal and tumor tissues from 16 patients with CRC to identify cancer-associated proteins and examine their roles in disease progression. Results: The analysis identified 10,329 proteins, including 531 cancer-associated proteins from the Catalogue Of Somatic Mutations In Cancer (COSMIC) database, and 48 proteins specifically linked to CRC. Notably, clusters of proteins showed consistent increases or decreases in abundance across disease stages, suggesting their roles in tumorigenesis and progression. Conclusions: Our findings suggest that proteome abundance trends may contribute to the identification of biomarker candidates and therapeutic targets in colorectal cancer. However, given the limited sample size and lack of subtype stratification, further studies using larger, statistically powered cohorts are warranted to establish clinical relevance. These proteins may provide insights into drug resistance and tumor heterogeneity. Limitations of the study include the inability to detect low-abundance proteins and reliance on protein abundance rather than functional activity. Future complementary approaches, such as affinity proteomics, are suggested to address these limitations.
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Open AccessReview
Uncovering Enzyme-Specific Post-Translational Modifications: An Overview of Current Methods
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Nashira H. Ridgeway and Kyle K. Biggar
Proteomes 2025, 13(3), 37; https://doi.org/10.3390/proteomes13030037 - 11 Aug 2025
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Post-translational modifications (PTMs) govern a multitude of protein functions within the cell, surpassing the basic function(s) encoded directly within the amino acid sequence. Despite the historical discovery of PTMs dating back over a century, recent technological advancements have facilitated the rapid expansion of
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Post-translational modifications (PTMs) govern a multitude of protein functions within the cell, surpassing the basic function(s) encoded directly within the amino acid sequence. Despite the historical discovery of PTMs dating back over a century, recent technological advancements have facilitated the rapid expansion of the known PTM landscape. However, the elucidation of enzyme–substrate relationships responsible for PTMs, particularly for those less studied, remains a challenging endeavor. This review provides an extensive overview of methods employed in the discovery of enzyme-specific substrates for PTM catalysis. Beginning with traditional experimental approaches rooted in chemistry, biochemistry and cell biology, this review progresses to recently developed computational strategies tailored for identifying enzyme–substrate interactions. The analysis reflects on the remarkable progress achieved in PTM research to date, underscoring the increasing role of computational and high-throughput techniques in expediting enzyme–substrate discovery. Furthermore, it highlights the potential of artificial intelligence to revolutionize PTM research and emphasizes the importance of unbiased high-throughput analysis in advancing our understanding of PTM networks. Ultimately, the review advocates for the integration of sophisticated computational strategies with experimental techniques to unravel the complex enzyme–substrate networks governing PTM-mediated cellular processes.
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Open AccessArticle
Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer
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Faris Alanazi, Ammar Sharif, Melissa Kidd, Emma-Jayne Keevill, Vanesa Biolatti, Richard D. Unwin, Peter Hoskin, Ananya Choudhury, Tim A. D. Smith and Conrado G. Quiles
Proteomes 2025, 13(3), 36; https://doi.org/10.3390/proteomes13030036 - 5 Aug 2025
Abstract
Background Plasma membrane proteins (PMPs) play key roles in cell signalling, adhesion, and trafficking, and are attractive therapeutic targets in cancer due to their surface accessibility. However, their typically low abundance limits detection by conventional proteomic approaches. Methods: To improve PMP detection, we
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Background Plasma membrane proteins (PMPs) play key roles in cell signalling, adhesion, and trafficking, and are attractive therapeutic targets in cancer due to their surface accessibility. However, their typically low abundance limits detection by conventional proteomic approaches. Methods: To improve PMP detection, we employed a surface proteomics workflow combining cell surface biotinylation and affinity purification prior to LC-MS/MS analysis in cervical (SiHa) and bladder (UMUC3) cancer cell lines cultured under normoxic (21% O2) or hypoxic (0.1% O2) conditions. Results: In SiHa cells, 43 hypoxia-upregulated proteins were identified exclusively in the biotin-enriched fraction, including ITGB2, ITGA7, AXL, MET, JAG2, and CAV1/CAV2. In UMUC3 cells, 32 unique upregulated PMPs were detected, including CD55, ADGRB1, SLC9A1, NECTIN3, and ACTG1. These proteins were not observed in corresponding whole-cell lysates and are associated with extracellular matrix remodelling, immune modulation, and ion transport. Biotinylation enhanced the detection of membrane-associated pathways such as ECM organisation, integrin signalling, and PI3K–Akt activation. Protein–protein interaction analysis revealed links between membrane receptors and intracellular stress regulators, including mitochondrial proteins. Conclusions: These findings demonstrate that surface biotinylation improves the sensitivity and selectivity of plasma membrane proteomics under hypoxia, revealing hypoxia-responsive proteins and pathways not captured by standard whole-cell analysis.
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(This article belongs to the Section Proteomics of Human Diseases and Their Treatments)
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Multiplex Targeted Proteomic Analysis of Cytokine Ratios for ICU Mortality in Severe COVID-19
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Rúben Araújo, Cristiana P. Von Rekowski, Tiago A. H. Fonseca, Cecília R. C. Calado, Luís Ramalhete and Luís Bento
Proteomes 2025, 13(3), 35; https://doi.org/10.3390/proteomes13030035 - 2 Aug 2025
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Background: Accurate and timely prediction of mortality in intensive care unit (ICU) patients, particularly those with COVID-19, remains clinically challenging due to complex immune responses. Proteomic cytokine profiling holds promise for refining mortality risk assessment. Methods: Serum samples from 89 ICU patients (55
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Background: Accurate and timely prediction of mortality in intensive care unit (ICU) patients, particularly those with COVID-19, remains clinically challenging due to complex immune responses. Proteomic cytokine profiling holds promise for refining mortality risk assessment. Methods: Serum samples from 89 ICU patients (55 discharged, 34 deceased) were analyzed using a multiplex 21-cytokine panel. Samples were stratified into three groups based on time from collection to outcome: ≤48 h (Group 1: Early), >48 h to ≤7 days (Group 2: Intermediate), and >7 days to ≤14 days (Group 3: Late). Cytokine levels, simple cytokine ratios, and previously unexplored complex ratios between pro- and anti-inflammatory cytokines were evaluated. Machine learning-based feature selection identified the most predictive ratios, with performance evaluated by area under the curve (AUC), sensitivity, and specificity. Results: Complex cytokine ratios demonstrated superior predictive accuracy compared to traditional severity markers (APACHE II, SAPS II, SOFA), individual cytokines, and simple ratios, effectively distinguishing discharged from deceased patients across all groups (AUC: 0.918–1.000; sensitivity: 0.826–1.000; specificity: 0.775–0.900). Conclusions: Multiplex cytokine profiling enhanced by computationally derived complex ratios may offer robust predictive capabilities for ICU mortality risk stratification, serving as a valuable tool for personalized prognosis in critical care.
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Open AccessArticle
Molecular Remodeling of the Sperm Proteome Following Varicocele Sclero-Embolization: Implications for Semen Quality Improvement
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Domenico Milardi, Edoardo Vergani, Francesca Mancini, Fiorella Di Nicuolo, Emanuela Teveroni, Emanuele Pierpaolo Vodola, Alessandro Oliva, Giuseppe Grande, Alessandro Cina, Roberto Iezzi, Michela Cicchinelli, Federica Iavarone, Silvia Baroni, Alberto Ferlin, Andrea Urbani and Alfredo Pontecorvi
Proteomes 2025, 13(3), 34; https://doi.org/10.3390/proteomes13030034 - 15 Jul 2025
Abstract
Background: Varicocele is a common condition involving the dilation of veins in the scrotum, often linked to male infertility and testicular dysfunction. This study aimed to elucidate the molecular effects of successful varicocele treatment on sperm proteomes following percutaneous sclero-embolization. Methods: High-resolution tandem
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Background: Varicocele is a common condition involving the dilation of veins in the scrotum, often linked to male infertility and testicular dysfunction. This study aimed to elucidate the molecular effects of successful varicocele treatment on sperm proteomes following percutaneous sclero-embolization. Methods: High-resolution tandem mass spectrometry was performed for proteomic profiling of pooled sperm lysates from five patients exhibiting improved semen parameters before and after (3 and 6 months) varicocele sclero-embolization. Data were validated by Western blot analysis. Results: Seven proteins were found exclusively in varicocele patients before surgery—such as stathmin, IFT20, selenide, and ADAM21—linked to inflammation and oxidative stress. After sclero-embolization, 55 new proteins emerged, including antioxidant enzymes like selenoprotein P and GPX3. Thioredoxin (TXN) and peroxiredoxin (PRDX3) were upregulated, indicating restoration of key antioxidant pathways. Additionally, the downregulation of some histones and the autophagy-related protein ATG9A suggests a shift toward an improved chromatin organization and a healthier cellular environment post-treatment. Conclusions: Varicocele treatment that improves sperm quality and fertility parameters leads to significant proteome modulation. These changes include reduced oxidative stress and broadly restored sperm maturation. Despite the limited patient cohort analyzed, these preliminary findings provide valuable insights into how varicocele treatment might enhance male fertility and suggest potential biomarkers for improved male infertility treatment strategies.
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(This article belongs to the Section Proteomics of Human Diseases and Their Treatments)
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Open AccessArticle
Comprehensive Integrated Analyses of Proteins and Metabolites in Equine Seminal Plasma (Horses and Donkeys)
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Xin Wen, Gerelchimeg Bou, Qianqian He, Qi Liu, Minna Yi and Hong Ren
Proteomes 2025, 13(3), 33; https://doi.org/10.3390/proteomes13030033 - 4 Jul 2025
Abstract
Background: The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify
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Background: The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify proteins and metabolites associated with sperm quality and reproductive ability in equine species. Methods: We utilized the CEROS instrument to assess the morphology and motility of sperm samples from three horses and three donkeys. Additionally, we statistically analyzed the mating frequency and pregnancy rates in both species. Meanwhile, the 4D-DIA high-throughput proteomic and metabolomic profiling of seminal plasma samples from horses and donkeys revealed a complex landscape of proteins and metabolites. Results: Our findings reveal a certain degree of correlation between seminal plasma proteins and metabolites and sperm quality, as well as overall fertility. Notably, we found that the proteins B3GAT3, XYLT2, CHST14, HS2ST1, GLCE, and HSPG2 in the glycosaminoglycan biosynthesis signaling pathway; the metabolites D-glucose, 4-phosphopantetheine, and 4-hydroxyphenylpyruvic acid in the tyrosine metabolism, starch, and source metabolisms; and pantothenate CoA biosynthesis metabolism present unique characteristics in the seminal plasma of equine species. Conclusions: This comprehensive approach provides new insights into the molecular mechanisms underlying sperm quality and has identified potential proteins and metabolites that could be used to indicate reproduction ability. The findings from this study could be instrumental in developing novel strategies to enhance equine breeding practices and reproductive management. Future research will focus on exploring their potential for clinical application in the equine industry.
Full article
(This article belongs to the Section Animal Proteomics)
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Open AccessArticle
Proteomic Profiling Reveals Novel Molecular Insights into Dysregulated Proteins in Established Cases of Rheumatoid Arthritis
by
Afshan Masood, Hicham Benabdelkamel, Assim A. Alfadda, Abdurhman S. Alarfaj, Amina Fallata, Salini Scaria Joy, Maha Al Mogren, Anas M. Abdel Rahman and Mohamed Siaj
Proteomes 2025, 13(3), 32; https://doi.org/10.3390/proteomes13030032 - 4 Jul 2025
Cited by 1
Abstract
Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder that predominantly affects synovial joints, leading to inflammation, pain, and progressive joint damage. Despite therapeutic advancements, the molecular basis of established RA remains poorly defined. Methods: In this study, we conducted an untargeted
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Background: Rheumatoid arthritis (RA) is a chronic autoimmune disorder that predominantly affects synovial joints, leading to inflammation, pain, and progressive joint damage. Despite therapeutic advancements, the molecular basis of established RA remains poorly defined. Methods: In this study, we conducted an untargeted plasma proteomic analysis using two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in samples from RA patients and healthy controls in the discovery phase. Results: Significantly (ANOVA, p ≤ 0.05, fold change > 1.5) differentially abundant proteins (DAPs) were identified. Notably, upregulated proteins included mitochondrial dicarboxylate carrier, hemopexin, and 28S ribosomal protein S18c, while CCDC124, osteocalcin, apolipoproteins A-I and A-IV, and haptoglobin were downregulated. Receiver operating characteristic (ROC) analysis identified CCDC124, osteocalcin, and metallothionein-2 with high diagnostic potential (AUC = 0.98). Proteins with the highest selected frequency were quantitatively verified by multiple reaction monitoring (MRM) analysis in the validation cohort. Bioinformatic analysis using Ingenuity Pathway Analysis (IPA) revealed the underlying molecular pathways and key interaction networks involved STAT1, TNF, and CD40. These central nodes were associated with immune regulation, cell-to-cell signaling, and hematological system development. Conclusions: Our combined proteomic and bioinformatic approaches underscore the involvement of dysregulated immune pathways in RA pathogenesis and highlight potential diagnostic biomarkers. The utility of these markers needs to be evaluated in further studies and in a larger cohort of patients.
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(This article belongs to the Special Issue Proteomics in Chronic Diseases: Issues and Challenges)
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Open AccessArticle
Comparative Label-Based Proteomics of Venoms from Echis ocellatus, Naja nigricollis, and Bitis arietans
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Abdulbaki Alfa-Ibrahim Adio, Samuel Odo Uko, Jiddah Muhammad Lawal, Ibrahim Malami, Nafiu Lawal, Amina Jega Yusuf Jega, Bilyaminu Abubakar, Muhammad Bashir Bello, Kasimu Ghandi Ibrahim, Murtala Bello Abubakar, Abdussamad Muhammad Abdussamad, Mujtaba Sulaiman Abubakar and Mustapha Umar Imam
Proteomes 2025, 13(3), 31; https://doi.org/10.3390/proteomes13030031 - 2 Jul 2025
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Background: Snake envenomation is a major public health issue in Nigeria, primarily due to bites from Echis ocellatus, Naja nigricollis, and Bitis arietans. Understanding their venom composition is essential for effective antivenom development. This study characterizes and compares the venom proteomes
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Background: Snake envenomation is a major public health issue in Nigeria, primarily due to bites from Echis ocellatus, Naja nigricollis, and Bitis arietans. Understanding their venom composition is essential for effective antivenom development. This study characterizes and compares the venom proteomes of these snakes using iTRAQ-based proteomics, focusing on key toxin families and their relative abundances. Methods: Venom samples were ethically collected from adult snakes, pooled by species, lyophilized, and stored for proteomic analysis. Proteins were extracted, digested with trypsin, and labeled with iTRAQ. Peptides were analyzed via mass spectrometry, and data were processed using Mascot and IQuant for protein identification and quantification. Results: E. ocellatus and B. arietans venoms had similar profiles, rich in C-type lectins, serine proteases, and phospholipase A2s. These comprised 17%, 11%, and 5% in E. ocellatus and 47%, 10%, and 7% in B. arietans, with metalloproteinases dominating both (53% and 47%). In N. nigricollis, three-finger toxins (9%) were most abundant, followed by metalloproteinases (3%). All species shared four core protein families, with N. nigricollis also containing four uncharacterized proteins. Conclusions: This study highlights venom compositional differences, advancing snake venom biology and informing targeted antivenom development.
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SDS Depletion from Intact Membrane Proteins by KCl Precipitation Ahead of Mass Spectrometry Analysis
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Tania Iranpour, Mapenzi Mirimba, Chloe Shenouda, Adam Lynch and Alan A. Doucette
Proteomes 2025, 13(3), 30; https://doi.org/10.3390/proteomes13030030 - 2 Jul 2025
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Background: Membrane proteins are preferentially solubilized with sodium dodecyl sulfate (SDS), which necessitates a purification protocol to deplete the surfactant prior to mass spectrometry analysis. However, maintaining solubility of intact membrane proteins is challenged in an SDS-free environment. SDS precipitation with potassium salts
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Background: Membrane proteins are preferentially solubilized with sodium dodecyl sulfate (SDS), which necessitates a purification protocol to deplete the surfactant prior to mass spectrometry analysis. However, maintaining solubility of intact membrane proteins is challenged in an SDS-free environment. SDS precipitation with potassium salts (KCl) offers a potentially viable workflow to deplete SDS and permit proteoform analysis. The purpose of this study is to devise a robust detergent-based protocol applicable for processing and analysis of intact membrane-associated proteoforms. Methods: The precipitation conditions impacting SDS removal from spinach chloroplasts and liver membrane proteome preparations were evaluated, capitalizing on optimization of pH (highly basic), addition of MS-compatible solubilizing additives (urea) and adjustment of the KCl to SDS ratio to maximize recovery and purity. Results: Characterization of the SDS-solubilized, KCl-precipitated spinach membrane preparation revealed multiple charge envelope MS spectra displaying high signal to noise, free of SDS adducts. Precipitation at pH 12 or with urea improved protein recovery and purity. Bottom-up analysis identified 1826 distinct liver protein groups from four independent SDS precipitation conditions. While precipitation at pH 8 without urea revealed a greater number of protein identifications by mass spectrometry, precipitation under highly basic conditions (pH 12) with urea provided higher membrane protein recovery and achieved the greatest number (732 of 1056) and largest percentage (69.3%) of membrane proteins identified in the SDS removal workflow. Conclusion: This workflow provides new opportunities for MS-based proteoform analysis by capitalizing on the benefits of SDS for protein extraction while maintaining high solubility and purity of intact proteins though KCl precipitation of the surfactant.
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Alterations in Tear Proteomes of Adults with Pre-Diabetes and Type 2 Diabetes Mellitus but Without Diabetic Retinopathy
by
Guoting Qin, Cecilia Chao, Shara Duong, Jennyffer Smith, Hong Lin, Wendy W. Harrison and Chengzhi Cai
Proteomes 2025, 13(3), 29; https://doi.org/10.3390/proteomes13030029 - 1 Jul 2025
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Background: Type 2 diabetes mellitus (T2DM) is an epidemic chronic disease that affects millions of people worldwide. This study aims to explore the impact of T2DM on the tear proteome, specifically investigating whether alterations occur before the development of diabetic retinopathy. Methods: Flush
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Background: Type 2 diabetes mellitus (T2DM) is an epidemic chronic disease that affects millions of people worldwide. This study aims to explore the impact of T2DM on the tear proteome, specifically investigating whether alterations occur before the development of diabetic retinopathy. Methods: Flush tear samples were collected from healthy subjects and subjects with preDM and T2DM. Tear proteins were processed and analyzed by mass spectrometry-based shotgun proteomics using a data-independent acquisition parallel acquisition serial fragmentation (diaPASEF) approach. Machine learning algorithms, including random forest, lasso regression, and support vector machine, and statistical tools were used to identify potential biomarkers. Results: Machine learning models identified 17 proteins with high importance in classification. Among these, five proteins (cystatin-S, S100-A11, submaxillary gland androgen-regulated protein 3B, immunoglobulin lambda variable 3–25, and lambda constant 3) exhibited differential abundance across these three groups. No correlations were identified between proteins and clinical assessments of the ocular surface. Notably, the 17 important proteins showed superior prediction accuracy in distinguishing all three groups (healthy, preDM, and T2DM) compared to the five proteins that were statistically significant. Conclusions: Alterations in the tear proteome profile were observed in adults with preDM and T2DM before the clinical diagnosis of ocular abnormality, including retinopathy.
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Evaluating Protein Extraction Techniques for Elucidating Proteomic Changes in Yeast Deletion Strains
by
Valentina Rossio and Joao A. Paulo
Proteomes 2025, 13(3), 28; https://doi.org/10.3390/proteomes13030028 - 1 Jul 2025
Abstract
Background: Alterations in protein abundance profiles in yeast deletion strains are frequently utilized to gain insights into cellular functions and regulatory networks, most of which are conserved in higher eukaryotes. Methods: This study investigates the impact of protein extraction methodologies on the whole
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Background: Alterations in protein abundance profiles in yeast deletion strains are frequently utilized to gain insights into cellular functions and regulatory networks, most of which are conserved in higher eukaryotes. Methods: This study investigates the impact of protein extraction methodologies on the whole proteome analysis of S. cerevisiae, comparing detergent-based lysis versus mechanical lysis with silica beads. We evaluated the proteomic profiles of wild-type and two yeast deletion strains, siz1Δ and nfi1Δ (siz2Δ), which are SUMO E3 ligases. Combining isobaric TMTpro-labeling with mass spectrometry using real-time search MS3, we profiled over 4700 proteins, covering approximately 80% of the yeast proteome. Results: Hierarchical clustering and principal component analyses revealed that the choice of protein extraction method significantly influenced the proteomic data, overshadowing the genetic variances among these strains. Notably, the detergent-based lysis showed superior performance in extracting proteins compared to mechanical lysis. Despite minimal proteomic alterations among strains, we observed consistent changes regardless of the lysis strategy in proteins such as Ino1, Rep1, Rep2, Snz1, and Fdh1 in both SUMO E3 ligase deletion strains, implying potential redundant mechanisms of control for these proteins. Conclusion: These findings underscore the importance of method selection at each step of sample preparation in proteomic studies and enhance our comprehension of cellular adaptations to genetic perturbations.
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(This article belongs to the Section Proteomics Technology and Methodology Development)
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Proteoform Patterns in Hepatocellular Carcinoma Tissues: Aspects of Oncomarkers
by
Elena Zorina, Natalia Ronzhina, Olga Legina, Nikolai Klopov, Victor Zgoda and Stanislav Naryzhny
Proteomes 2025, 13(3), 27; https://doi.org/10.3390/proteomes13030027 - 1 Jul 2025
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Background: Human proteins exist in numerous modifications—proteoforms—which are promising targets for biomarker studies. In this study, we aimed to generate comparative proteomics data, including proteoform patterns, from hepatocellular carcinoma (HCC) and nonmalignant liver tissues. Methods: To investigate protein profiles and proteoform patterns, we
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Background: Human proteins exist in numerous modifications—proteoforms—which are promising targets for biomarker studies. In this study, we aimed to generate comparative proteomics data, including proteoform patterns, from hepatocellular carcinoma (HCC) and nonmalignant liver tissues. Methods: To investigate protein profiles and proteoform patterns, we employed a panoramic, integrative top-down proteomics approach: two-dimensional gel electrophoresis (2DE) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Results: We visualized over 2500 proteoform patterns per sample type, enabling the identification of distinct protein signatures and common patterns differentiating nonmalignant and malignant liver cells. Among these, 1270 protein patterns were uniformly observed across all samples. Additionally, 38 proteins—including pyruvate kinase PKM (KPYM), annexin A2 (ANXA2), and others—exhibited pronounced differences in proteoform patterns between nonmalignant and malignant tissues. Conclusions: Most proteoform patterns of the same protein were highly similar, with the dominant peak corresponding to theoretical (unmodified) protein parameters. However, certain proteins displayed altered proteoform patterns and additional proteoforms in cancer compared to controls. These proteins were prioritized for further characterization.
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