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Proteomes
Volume 13
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Proteomes, Volume 13, Issue 3 (September 2025) – 18 articles

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15 pages, 1493 KB  
Article
Proteomic Analysis of Sputum from Patients with Active Tuberculosis
by 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
Abstract
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. [...] Read more.
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. Full article
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13 pages, 3545 KB  
Article
Proteomic Analysis of the Periodontal Ligament During Orthodontic Movement: A Study in Rats
by 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
Abstract
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 [...] Read more.
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. Full article
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27 pages, 1868 KB  
Article
Bidirectional Interaction Between PGE2-Preconditioned Mesenchymal Stem Cells and Myofibroblasts Mediates Anti-Fibrotic Effects: A Proteomic Investigation into Equine Endometrial Fibrosis Reversal
by 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
Viewed by 309
Abstract
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 [...] Read more.
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. Full article
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15 pages, 1799 KB  
Article
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
by Malte Bayer, Michael Snyder and Simone König
Proteomes 2025, 13(3), 40; https://doi.org/10.3390/proteomes13030040 - 27 Aug 2025
Viewed by 392
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 [...] Read more.
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. Full article
(This article belongs to the Section Proteomics Technology and Methodology Development)
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29 pages, 3108 KB  
Article
Adiposome Proteomics Uncover Molecular Signatures of Cardiometabolic Risk in Obese Individuals
by 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
Viewed by 651
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Proteomics in Chronic Diseases: Issues and Challenges)
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13 pages, 2308 KB  
Article
Identification of Cancer-Associated Proteins in Colorectal Cancer Using Mass Spectrometry
by 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
Viewed by 475
Abstract
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 [...] Read more.
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. Full article
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17 pages, 1488 KB  
Review
Uncovering Enzyme-Specific Post-Translational Modifications: An Overview of Current Methods
by Nashira H. Ridgeway and Kyle K. Biggar
Proteomes 2025, 13(3), 37; https://doi.org/10.3390/proteomes13030037 - 11 Aug 2025
Viewed by 549
Abstract
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 [...] Read more.
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. Full article
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18 pages, 2229 KB  
Article
Cell Surface Proteomics Reveals Hypoxia-Regulated Pathways in Cervical and Bladder Cancer
by 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
Viewed by 782
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 [...] Read more.
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. Full article
(This article belongs to the Section Proteomics of Human Diseases and Their Treatments)
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48 pages, 4602 KB  
Article
Multiplex Targeted Proteomic Analysis of Cytokine Ratios for ICU Mortality in Severe COVID-19
by 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
Viewed by 635
Abstract
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 [...] Read more.
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. Full article
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19 pages, 42627 KB  
Article
Molecular Remodeling of the Sperm Proteome Following Varicocele Sclero-Embolization: Implications for Semen Quality Improvement
by 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
Viewed by 729
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 [...] Read more.
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. Full article
(This article belongs to the Section Proteomics of Human Diseases and Their Treatments)
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21 pages, 1482 KB  
Article
Comprehensive Integrated Analyses of Proteins and Metabolites in Equine Seminal Plasma (Horses and Donkeys)
by 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
Viewed by 782
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 [...] Read more.
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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24 pages, 4271 KB  
Article
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
Viewed by 954
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Proteomics in Chronic Diseases: Issues and Challenges)
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21 pages, 1637 KB  
Article
Comparative Label-Based Proteomics of Venoms from Echis ocellatus, Naja nigricollis, and Bitis arietans
by 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
Viewed by 1574
Abstract
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 [...] Read more.
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. Full article
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21 pages, 3424 KB  
Article
SDS Depletion from Intact Membrane Proteins by KCl Precipitation Ahead of Mass Spectrometry Analysis
by 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
Viewed by 852
Abstract
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 [...] Read more.
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. Full article
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16 pages, 2558 KB  
Article
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
Viewed by 648
Abstract
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 [...] Read more.
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. Full article
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15 pages, 2565 KB  
Article
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
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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 [...] Read more.
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. Full article
(This article belongs to the Section Proteomics Technology and Methodology Development)
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16 pages, 6224 KB  
Article
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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Abstract
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 [...] Read more.
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. Full article
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27 pages, 2972 KB  
Review
Next-Generation Protein–Ligand Interaction Networks: APEX as a Powerful Technology
by José Miguel Quintero-Ferrer, Lucas Silva de Oliveira, Paula Marian Vieira Goulart, Thiago Albuquerque Souza Campos, Coralie Martin, Philippe Grellier, Izabela Marques Dourado Bastos and Sébastien Charneau
Proteomes 2025, 13(3), 26; https://doi.org/10.3390/proteomes13030026 - 23 Jun 2025
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Abstract
Peroxidases are essential enzymes that catalyze redox reactions, with wide-ranging biological implications. Among these, an enhanced ascorbate peroxidase (APEX) has emerged as a valuable tool for studying intricate intracellular events with spatiotemporal precision, particularly in protein–protein, protein–RNA, and protein–DNA interaction networks in living [...] Read more.
Peroxidases are essential enzymes that catalyze redox reactions, with wide-ranging biological implications. Among these, an enhanced ascorbate peroxidase (APEX) has emerged as a valuable tool for studying intricate intracellular events with spatiotemporal precision, particularly in protein–protein, protein–RNA, and protein–DNA interaction networks in living cells. This review discusses APEX’s structural and functional attributes, its evolution through genetic engineering, and its transformative applications in high-resolution mapping used for proteomic and transcriptomic studies. Furthermore, it highlights recent advancements in substrate innovation and addresses current challenges and future directions in leveraging APEX for cutting-edge biological research. Full article
(This article belongs to the Section Spatio-Temporal Proteomics)
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