Proteomes

13 pages, 3418 KB

Open AccessArticle

A Dual-Background Statistical Framework for Phosphoproteomics Highlights Intrinsic, High-Confidence Phosphorylation Signature by Mitigating Orthogonal Sources of Bias

by Bin Deng

Proteomes 2026, 14(3), 33; https://doi.org/10.3390/proteomes14030033 - 7 Jul 2026

Abstract

Background: Distinguishing genuine kinase–substrate motifs from background noise is a growing challenge, as mass spectrometry (MS)-based global phosphoproteomics identifies a rapidly expanding set of phosphorylation sites. One of the major limitations is selecting an appropriate background model that systematically controls both technical and [...] Read more.

Background: Distinguishing genuine kinase–substrate motifs from background noise is a growing challenge, as mass spectrometry (MS)-based global phosphoproteomics identifies a rapidly expanding set of phosphorylation sites. One of the major limitations is selecting an appropriate background model that systematically controls both technical and biological sources of bias. Although using the entire proteome as a background in a FASTA format considers the overall amino acid composition, it is still prone to biases from protein abundance and the uneven distribution of sequence space (particularly around low-abundance proteins). By contrast, internal background methods can control experiment-specific detection biases, but they may not fully capture residue-specific compositions or general trends in phosphorylation. Methods: I develop a Dual-Background Enrichment (DBE) framework with a position-specific enrichment (PSE) strategy, which involves analyzing motif enrichment against two distinct background models: (1) A residue-heterogeneous internal background composed of phospho-motifs centered on the residue; e.g., phosphoserine (pS) motifs are tested relative to the pool of all detected phosphothreonine (pT) and phosphotyrosine (pY) motifs from the same experiment. (2) A FASTA background that includes all S, T, and Y residues in the UniProtKB proteome sequences. Results: Motifs are classified as high confidence if they meet statistical significance (q ≤ 0.05, fold enrichment > 1.5) against both background models. Conclusion: By applying the DBE strategy to a large-scale phosphoproteomics dataset, we distinguish motifs driven by amino acid composition (enriched in FASTA background only) from those reflecting kinase substrate specificity (enriched in both backgrounds). This dual-reference approach reduces false positives arising from sequence composition bias and enriches high-confidence candidate kinase recognition motifs. Full article

(This article belongs to the Section Proteome Bioinformatics)

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24 pages, 1650 KB

Open AccessReview

A One Health Framework for Proteomics Across the Tree of Life to Advance Food Security, Animal Health, and Ecosystem Resilience

by Tarun Mishra, Ritudhwaj Tiwari, Tuyelee Das and Maneesh Lingwan

Proteomes 2026, 14(3), 32; https://doi.org/10.3390/proteomes14030032 - 24 Jun 2026

Abstract

As global ecosystems and food systems face unprecedented anthropogenic and climatic challenges, there is a demand for an integrated understanding of biological systems. Proteomics has emerged as a definitive approach offering a direct view of the molecular phenotype, yet it is traditionally separated [...] Read more.

As global ecosystems and food systems face unprecedented anthropogenic and climatic challenges, there is a demand for an integrated understanding of biological systems. Proteomics has emerged as a definitive approach offering a direct view of the molecular phenotype, yet it is traditionally separated into plant and animal disciplines. With recent advances in mass spectrometry (MS) and bioinformatics tools, this prospective review proposes that combining a One Health proteomics approach with deep-learning data analysis can revolutionize global food security, animal productivity, and ecosystem health by uncovering proteoform signatures that drive resilience across life. The potential of a unified One Health proteomic framework, highlighting major developments, including 4D proteomics, Data-Independent Acquisition (DIA), and single-cell resolution, and emphasizes their capacity to resolve the complex proteoform landscape across kingdoms. Review emphasizes the applications of proteogenomics as a cross-disciplinary tool to improve genome annotations, explain evolutionary differences, discover biomarkers in animals and resolve complex signaling networks in plants under stress. Nevertheless, contemporary proteogenomics methods still show limitations in their ability to comprehensively resolve proteoforms due to the fact that the use of peptide-based approaches makes it difficult to fully appreciate the post-translational modifications specific to each protein isoform. We show that One Health proteomics will provide a transformative roadmap for deciphering the functional proteoform signatures that underpin resilience across the tree of life. Full article

(This article belongs to the Special Issue Plant Genomics and Proteomics)

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17 pages, 1902 KB

Open AccessArticle

Nuclear Proteomics to Understand the Promotive Effect of Plant-Derived Smoke Solution on Wheat Under Salt Stress

by Sheikh Shohag, Hisateru Yamaguchi, Keisuke Hitachi, Kunihiro Tsuchida, Shafiq Ur Rehman and Setsuko Komatsu

Proteomes 2026, 14(2), 31; https://doi.org/10.3390/proteomes14020031 - 15 Jun 2026

Abstract

Background: Salinity, which hampers wheat growth and development, is one of the major abiotic stresses. Plant-derived smoke (PDS) solution alleviates salt stress and promotes wheat growth and development; however, the underlying molecular mechanisms have not been completely clarified. Methods: In this study, nuclear [...] Read more.

Background: Salinity, which hampers wheat growth and development, is one of the major abiotic stresses. Plant-derived smoke (PDS) solution alleviates salt stress and promotes wheat growth and development; however, the underlying molecular mechanisms have not been completely clarified. Methods: In this study, nuclear proteomics was employed to reveal the promotive effect of PDS solution on salt-stressed wheat. Nuclear fractions were isolated from wheat roots, and their purity was confirmed via enrichment of histone H3 and reduction of cytosolic ascorbate peroxidase. Using this nuclear purification technique, label-free nano LC–MS/MS-based nuclear proteomics was performed to identify differentially abundant nuclear proteins in salt-stressed wheat with or without PDS solution treatment. Results: Salt stress decreased histone H2A and DNA polymerase levels, whereas PDS solution treatment of salt-stressed wheat increased levels of histone variants (H2A, H2B, H3, and H4), DNA polymerase, and DNA topoisomerase II. In addition, the PDS solution increased the levels of pre-mRNA cleavage factor Im 25 kDa subunit and RNA helicase in salt-stressed wheat. Immunoblot analysis further validated the increase in histone deacetylase levels triggered by the PDS solution treatment in the salt-stressed wheat. Conclusions: These results suggest that PDS solution alters nuclear proteins in a way that contributes to chromatin remodeling and transcription during salt stress. Full article

(This article belongs to the Special Issue Plant Genomics and Proteomics)

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20 pages, 3043 KB

Open AccessArticle

Development of a Xylene-Free Sample Preparation Protocol for Quantitative Proteomics of Clinically Relevant Formaldehyde-Fixed Paraffin-Embedded Needle Biopsy Samples

by Gontse Mabuse Moagi, Lívia Beke, Gábor Méhes, Gábor Kecskeméti, Zoltán Szabó, Lilla Turiák and Éva Csősz

Proteomes 2026, 14(2), 30; https://doi.org/10.3390/proteomes14020030 - 14 Jun 2026

Abstract

Background: Fresh frozen tissues are considered the gold standard for proteomic analyses due to their superior preservation of protein integrity; however, their use is limited by the logistical and financial requirements of long-term cold storage. Formaldehyde-fixed paraffin-embedded (FFPE) tissues provide a practical alternative, [...] Read more.

Background: Fresh frozen tissues are considered the gold standard for proteomic analyses due to their superior preservation of protein integrity; however, their use is limited by the logistical and financial requirements of long-term cold storage. Formaldehyde-fixed paraffin-embedded (FFPE) tissues provide a practical alternative, owing to their stability and widespread availability in clinical settings. A critical step in FFPE proteomics is deparaffinization, which traditionally relies on organic solvents such as xylene, along with the efficient reversal of formaldehyde-induced crosslinks. Methods: In this study, we evaluated multiple FFPE protein extraction and digestion workflows including chaotropic, surfactant-based, and detergent-free approaches in combination with xylene-free deparaffinization strategies, using label-free data-independent acquisition (DIA) LC-MS/MS. Results: Among the tested methods, a chaotropic, reductant, and surfactant-free in-solution digestion workflow demonstrated robust protein and peptide recovery. A modified version of this protocol further improved peptide coverage while maintaining comparable protein depth. The applicability of the optimized workflow was assessed using FFPE needle biopsy samples from control, hepatic steatosis, and liver fibrosis groups. Exploratory proteomic patterns were observed across conditions, with hepatic steatosis associated with early activation of stress-response pathways, while fibrosis showed evidence suggesting altered lipid metabolism. Conclusions: Overall, this study presents a simple, xylene-free, and MS-compatible workflow for FFPE proteomics that is suitable for low-input clinical samples and may support broader application of archival tissues in proteomic research. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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19 pages, 1412 KB

Open AccessSystematic Review

Systematic Review of Protein Signatures for Clinical Monitoring of Osteonecrosis of the Jaw: Meta-Analysis and Insights from Bioinformatics-Driven Proteomics

by Helena Oliveira Deróbio, Isabela dos Reis Souza, François Isnaldo Dias Caldeira, Fernanda Gonçalves Basso and Taisa Nogueira Pansani

Proteomes 2026, 14(2), 29; https://doi.org/10.3390/proteomes14020029 - 10 Jun 2026

Abstract

Background: Several studies have investigated the clinical and immunological aspects of medication-related osteonecrosis of the jaw (MRONJ). However, the underlying immunological mechanisms and signaling pathways involved in its pathophysiology remain incompletely understood. This systematic review and meta-analysis, complemented by bioinformatics analyses, aimed to [...] Read more.

Background: Several studies have investigated the clinical and immunological aspects of medication-related osteonecrosis of the jaw (MRONJ). However, the underlying immunological mechanisms and signaling pathways involved in its pathophysiology remain incompletely understood. This systematic review and meta-analysis, complemented by bioinformatics analyses, aimed to identify proteomic biomarkers associated with MRONJ. Methods: Six databases (PubMed, Embase, Scopus, Web of Science, Cochrane Library, and VHL) were searched, along with gray literature and manual searches. Observational studies in English comparing proteomic profiles of individuals with and without MRONJ were included. Study selection and data management were conducted using EndNote™ X8 and Rayyan.ai, and risk of bias was assessed using the QUADOMICS tool. Functional enrichment analysis was performed using g:Profiler and Reactome, and interaction networks were constructed using GeneMANIA, STRING, and MetaboAnalyst (Cytoscape program; version 3.10.1). Meta-analysis was performed in RStudio (R-4.5, Rstudio extension 2025.05.1+513) (α = 0.05). Results: Three studies were included in the review, and two in the meta-analysis. The meta-analysis showed higher salivary levels of Apolipoprotein B-100 (APOB), Apolipoprotein A-II (APOA2), and Heparin Cofactor 2 (SERPIND1) in MRONJ patients, while the protein Keratin (KRT16) showed reduced levels without statistical significance. Bioinformatics analyses indicated involvement in lipid metabolism, impaired tissue repair, and inflammatory and immune responses. Conclusions: These findings suggest altered salivary proteomic signatures in MRONJ for APOB, APOA2, SERPIND1, and KRT16 proteins. Full article

(This article belongs to the Section Identification of Potential Biomarkers and Potential Therapeutic Targets)

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34 pages, 12216 KB

Open AccessReview

Diverse Forms of Autophagy and Their Roles in Liver Disease and Aging: A Comprehensive Review

by Seoyoon Heo, Min Young Lee, Che Yeon Jeong, Dong Ha Kim and Ji Hye Jun

Proteomes 2026, 14(2), 28; https://doi.org/10.3390/proteomes14020028 - 27 May 2026

Abstract

The liver is a central metabolic organ that integrates nutrient sensing, lipid handling, and detoxification to maintain systemic homeostasis. In metabolic dysfunction–associated steatotic liver disease (MASLD), chronic metabolic overload accelerates hepatocyte senescence, impairing regenerative capacity and promoting progression toward fibrosis and hepatocellular carcinoma. [...] Read more.

The liver is a central metabolic organ that integrates nutrient sensing, lipid handling, and detoxification to maintain systemic homeostasis. In metabolic dysfunction–associated steatotic liver disease (MASLD), chronic metabolic overload accelerates hepatocyte senescence, impairing regenerative capacity and promoting progression toward fibrosis and hepatocellular carcinoma. While transcriptomic studies have provided important insights into stress-responsive pathways, they incompletely capture the proteome remodeling and proteoform-level alterations that govern hepatocyte function during aging and disease. Recent mass spectrometry–based proteomics studies have revealed that disruption of autophagy-dependent proteome homeostasis is a defining feature of senescent hepatocytes. Quantitative analyses demonstrate coordinated alterations in selective autophagy pathways—including lipophagy, mitophagy, ferritinophagy, ER-phagy, and pexophagy—accompanied by organelle-specific protein abundance signatures and remodeling of autophagy-related proteoforms. These findings position proteomics as an essential tool for resolving the spatial and functional reorganization of hepatocyte proteomes that cannot be inferred from transcript abundance alone. In this review, we synthesize proteomics-driven evidence defining selective autophagy dysfunction in aging and MASLD livers, critically evaluate methodological limitations, and propose a conceptual framework in which impaired selective autophagy acts as a proteome-level driver of hepatocyte senescence. We further outline future directions for proteoform-resolved and spatial proteomics approaches aimed at identifying actionable targets for therapeutic intervention in liver disease. Full article

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25 pages, 9441 KB

Open AccessArticle

Quantitative Metaproteomic Characterization of Acetic Acid Bacteria Reveals Functional Dynamics During Verdejo Wine Acetification

by Cristina Campos-Vázquez, Juan C. García-García, Juan Carbonero-Pacheco, Juan J. Román-Camacho, Roger Consuegra-Rivera, Teresa García-Martínez, Isidoro García-García, Inés M. Santos-Dueñas and Juan Carlos Mauricio

Proteomes 2026, 14(2), 27; https://doi.org/10.3390/proteomes14020027 - 20 May 2026

Abstract

Background: Acetification is a complex process driven by acetic acid bacteria (AAB), in which high ethanol and acidity levels require strong microbial metabolic adaptation. Although the microbiota involved in vinegar production has been described, the functional mechanisms that enable these bacteria to maintain [...] Read more.

Background: Acetification is a complex process driven by acetic acid bacteria (AAB), in which high ethanol and acidity levels require strong microbial metabolic adaptation. Although the microbiota involved in vinegar production has been described, the functional mechanisms that enable these bacteria to maintain metabolic activity remain poorly understood. In this study, the functional dynamics of AAB during Verdejo vinegar acetification were analyzed using a quantitative metaproteomic approach. Methods: Acetification was performed in submerged culture under semi-continuous conditions, and samples were collected at four stages of the cycle (S1–S4). Results: LC-MS/MS analysis led to the identification of 1626 proteins, of which 1409 were assigned to the Acetobacteraceae family. Komagataeibacter europaeus was the dominant species (73.7%). Hierarchical clustering revealed four protein abundance patterns, and differential analysis identified 350 proteins with increased abundance and 169 with decreased abundance, with the greatest changes observed between S1 and S4. Functional annotation and protein–protein interaction analyses indicated that the main metabolic adaptations involve pathways related to energy metabolism, amino acid biosynthesis, membrane-associated functions, cellular homeostasis, and acid stress response. Conclusions: Overall, the results show that K. europaeus concentrates most of the metabolic activity during acetification and that proteome reorganization reflects key molecular strategies for adaptation and survival under high-acidity conditions. Full article

(This article belongs to the Section Microbial Proteomics)

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13 pages, 840 KB

Open AccessPerspective

What Are the Practical Applications of Single-Cell Proteomics?

by Benjamin C. Orsburn

Proteomes 2026, 14(2), 26; https://doi.org/10.3390/proteomes14020026 - 18 May 2026

Abstract

Single-cell proteomics (SCP) is an exciting new field of study with developments in the areas of sample preparation, instrumentation and informatics. SCP has captured the imagination of biologists and clinicians and the critical interest of both academic and commercial mass-spectrometry groups. Currently (i.e., [...] Read more.

Single-cell proteomics (SCP) is an exciting new field of study with developments in the areas of sample preparation, instrumentation and informatics. SCP has captured the imagination of biologists and clinicians and the critical interest of both academic and commercial mass-spectrometry groups. Currently (i.e., at the time this manuscript was written), SCP is still difficult and slow relative to competing single-cell technologies. What SCP may lose in relative throughput, it trades for direct analysis of protein and proteoforms, albeit with biases toward those of the highest relative concentration in each cell. These strengths may not make SCP the technology of choice for every study. This perspective is intended to identify current and future biological or clinical areas where SCP has or could have the greatest potential to advance human health and knowledge. I will also discuss applications where SCP would be less impactful than other technologies and where SCP, when mature, could play a true role in clinical diagnostics. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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18 pages, 1800 KB

Open AccessArticle

Changes in the Proteome and Phosphoproteome of Zea mays Tissues in Drought Stress Show Plant Tissue Responses from Dehydrins, Carboxylic Acid Metabolism, RNA Splicing and Transcription Factors

by Georgina H. Charlton, Cleidiane Zampronio, Andrew R. Bottrill, John Sinclair, Peter M. Kilby and Alexandra M. E. Jones

Proteomes 2026, 14(2), 25; https://doi.org/10.3390/proteomes14020025 - 9 May 2026

Abstract

Background: Maize is a vital crop, supporting 19.5% of global calorie intake. However, maize is vulnerable to even brief periods of drought which substantially reduces seed set and therefore yield. Methods: To identify proteins involved in responses of maize to drought, soluble proteins [...] Read more.

Background: Maize is a vital crop, supporting 19.5% of global calorie intake. However, maize is vulnerable to even brief periods of drought which substantially reduces seed set and therefore yield. Methods: To identify proteins involved in responses of maize to drought, soluble proteins were extracted from leaf and silk tissues of Zea mays and protein abundance and phosphorylation status were quantified relative to well-watered controls. Label-free quantification and phosphopeptide enrichment were applied to the same biological samples and over 300 proteins were identified with significantly different changes. Results: Proteins known to be involved in drought responses were identified, such as the abscisic acid pathway and transcription factors. Of particular interest is a group of dehydrins quantified at both total protein and phosphopeptide levels, permitting insight into stoichiometry. The biological function of dehydrins in the model plant Arabidopsis thaliana is known to be regulated by phosphorylation. Conclusions: Translation of protein function from model plant to crops remains highly challenging because genome duplication has created complex sets of orthologous and homologous proteins. By focusing on proteomic changes during crop stress responses, this work enables the identification of known and novel proteins, substantially aiding the transfer of knowledge from model plants to crops. Full article

(This article belongs to the Special Issue Plant Genomics and Proteomics)

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15 pages, 1999 KB

Open AccessArticle

Cell Type-Specific Proteomic Cargo in Human Brain Endothelial, Astrocyte, and Neuronal Extracellular Vesicles

by Hope K. Hutson, Guoting Qin, Chengzhi Cai and Gergana G. Nestorova

Proteomes 2026, 14(2), 24; https://doi.org/10.3390/proteomes14020024 - 1 May 2026

Abstract

Background: Extracellular vesicles (EVs) mediate intercellular communication in the central nervous system and are a major source of biomarkers. This study characterizes the EV-derived proteome secreted by human endothelial brain cells (HEBCs), astrocytes, and neurons to identify cell-specific roles in intercellular communication in [...] Read more.

Background: Extracellular vesicles (EVs) mediate intercellular communication in the central nervous system and are a major source of biomarkers. This study characterizes the EV-derived proteome secreted by human endothelial brain cells (HEBCs), astrocytes, and neurons to identify cell-specific roles in intercellular communication in the brain. Methods: Mass spectrometry analyses of EVs and corresponding parent cells were performed to identify differentially enriched proteins. Gene Ontology (GO) analysis of statistically significant, abundantly expressed proteins between EVs and parent cells (log₂ fold-change ≥ 2.0, p < 0.05) was performed to assess cell-specific functions. Results: Proteome analysis identified on average 932 proteins in astrocyte EVs (versus 1725 in parent cells), 1040 in HEBC EVs (versus 5451 in parent cells), and 470 in neuronal EVs (versus 578 in parent cells). The analysis indicated that astrocytes had the highest number of significantly abundant proteins (118), followed by HEBCs (24) and neurons (25). Astrocyte EVs were enriched in lipoproteins, complement factors, and protease inhibitors; HEBCs EVs in tight junction proteins, adhesion molecules, and protease regulators; and neuronal EVs in chromatin-associated histones, tubulin isoforms, and RNA-binding proteins. Conclusions: The proteomic signatures of EVs from different neurovascular unit cells suggest specialized roles in blood–brain barrier homeostasis, immune regulation, and synaptic and epigenetic signaling under healthy conditions. These baseline signatures provide a framework for future studies to investigate how brain cell-derived EVs may contribute to neurodegenerative disorders. Full article

(This article belongs to the Section Extracellular Vesicles)

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20 pages, 4891 KB

Open AccessArticle

Dissection of Genotype-Dependent Responses Reveals Leaf Proteome Signatures Associated with Maize Thermotolerance During Flowering Under Enclosure-Imposed Heat Stress

by Ruixiang Liu, Xiaohang Li, Zixin Zha, Meijing Zhang, Lingjie Kong, Yakun Cui, Wenming Zhao, Qingchang Meng, Youhua Wang and Yanping Chen

Proteomes 2026, 14(2), 23; https://doi.org/10.3390/proteomes14020023 - 29 Apr 2026

Abstract

Background: During maize anthesis, heat stress severely limits productivity—particularly under humid conditions where high humidity suppresses transpirational cooling, forcing tissues to endure direct thermal load. Methods: Using field enclosures to impose enclosure-imposed humid heat shock (EHS), we screened 135 maize inbred lines for [...] Read more.

Background: During maize anthesis, heat stress severely limits productivity—particularly under humid conditions where high humidity suppresses transpirational cooling, forcing tissues to endure direct thermal load. Methods: Using field enclosures to impose enclosure-imposed humid heat shock (EHS), we screened 135 maize inbred lines for flowering-stage yield resilience, using grain weight per ear at maturity under EHS relative to the corresponding control (CK) condition as the primary selection criterion. Based on this screen, we selected two tolerant (R025, R100) and two sensitive (R133, R135) genotypes for data-independent acquisition mass spectrometry (DIA-MS) profiling of the tassel-subtending leaf. Results: At baseline, the selected tolerant lines exhibited a constitutively distinct proteomic state, including lower abundance of light-harvesting complex components and higher abundance or detection frequency of several regulatory proteins, including SRK2E/OST1 and HSF-B2a. Under sustained EHS, the selected sensitive lines showed extensive proteomic disruption, including reduced abundance of photosynthesis-related proteins and oxidative phosphorylation, together with increased abundance of proteins associated with endoplasmic reticulum stress responses and protein turnover. In contrast, the selected tolerant lines displayed a more constrained acclimation response, characterized by relative maintenance of photosynthesis-related proteins together with selective increases in chaperone systems (HSP90/sHSPs) and benzoxazinoid biosynthesis-related proteins. Several proteins showed switch-like detection patterns between the selected tolerant and sensitive lines, including TMEM97-like and a peptidyl-prolyl isomerase, indicating potentially distinct regulatory states. Conclusions: These findings suggest that tolerant performance under enclosure-imposed heat stress is associated with a pre-conditioned proteomic state and enhanced protein homeostasis (proteostasis) buffering capacity that may help preserve photosynthetic function during flowering-stage stress. The identified proteins should be regarded as candidate markers requiring further functional validation before any application in breeding programs aimed at improving adaptation to increasingly frequent heat-stress events. Full article

(This article belongs to the Special Issue Plant Genomics and Proteomics)

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3 pages, 682 KB

Open AccessEditorial

Proteomes Annual Report Card 2025

by Jens R. Coorssen and Matthew P. Padula

Proteomes 2026, 14(2), 22; https://doi.org/10.3390/proteomes14020022 - 24 Apr 2026

Abstract

We begin by expressing our sincere thanks to all Editorial Board Members, Guest Editors, Reviewers, Authors, and the staff in the Editorial Office for their dedicated service in support of Proteomes [...] Full article

28 pages, 2341 KB

Open AccessArticle

Prospective ICH Q2(R2)-Aligned Total-Error Validation of Label-Free Untargeted Proteomics for Host Cell Protein Quantification in Biotherapeutics

by Somar Khalil, Jean-François Dierick, Pascal Bourguignon and Michel Plisnier

Proteomes 2026, 14(2), 21; https://doi.org/10.3390/proteomes14020021 - 23 Apr 2026

Abstract

Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome [...] Read more.

Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20–80 ng) and analyzed in four independent assays (198 injections), supporting one-way random-effects ANOVA with Welch–Satterthwaite adjustment. Peptide-level identification error was evaluated by dual entrapment. Results: Empirical false-discovery proportions were below 1% at q = 0.01. Weighted least-squares regression (R² = 0.993) confirmed stable proportional compression with 81–85% recovery. Repeatability dominated the variance structure (median CV 2.7%); intermediate precision SD ranged from 0.69% to 3.81%. Both 95% β-expectation and 95/95 content tolerance intervals were contained within ±30% at all levels, defining a validated range of 20–80 ng. Abundance-stratified TE profiling revealed concentration-dependent calibration heterogeneity, with stratum-specific intervals within ±35% defining an abundance-aware LLOQ of 3.6 ppm (P95 = 3.87 ppm). Robustness under independent search software (FragPipe v24.0, CCC = 0.998) and cross-platform acquisition (Astral, CCC = 0.980) remained within ±30% limits. Conclusions: This constitutes the first prospective ICH Q2(R2)-aligned validation of untargeted proteomics for HCP quantification, with a transferable statistical framework for high-dimensional analytical methods. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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15 pages, 1002 KB

Open AccessReview

Enabling Next-Generation Mass Spectrometry-Based Proteomics: Standards, Proteoform Resolution, and FAIR, Reproducible, and Quantitative Analysis

by Rui Vitorino

Proteomes 2026, 14(2), 20; https://doi.org/10.3390/proteomes14020020 - 21 Apr 2026

Abstract

Recent advances in mass spectrometry, data-independent acquisition, proteoform-resolving workflows, and multi-omics integration have significantly expanded the scale and scope of proteomics. However, the reuse and translational application of these datasets are limited by inconsistent standards, insufficient metadata, and inadequate computational interoperability. Proteoform-centric approaches [...] Read more.

Recent advances in mass spectrometry, data-independent acquisition, proteoform-resolving workflows, and multi-omics integration have significantly expanded the scale and scope of proteomics. However, the reuse and translational application of these datasets are limited by inconsistent standards, insufficient metadata, and inadequate computational interoperability. Proteoform-centric approaches provide higher molecular resolution by capturing intact protein variants and patterns of post-translational modification. Computational methods, including selected applications of machine learning and large language models (LLMs), are increasingly used for tasks such as spectral prediction and pattern discovery in clinical proteomics datasets. Despite these advancements, FAIR (Findable, Accessible, Interoperable, and Reusable) data practices, proteoform biology, and AI analytics are often pursued independently. This work presents an integrated framework for next-generation proteomics in which standardization and FAIR (Findable, Accessible, Interoperable, and Reusable) principles establish machine-actionable foundations for proteoform-resolved analysis and computational inference. It examines community efforts to promote data sharing and interoperability, as well as strategies for characterizing proteoforms using bottom-up, middle-down, and top-down approaches. It also highlights emerging AI and ML applications within the proteomics workflow. The framework emphasizes the importance of treating proteoforms as primary computational entities and adopting FAIR practices during data collection to enable reproducible and interpretable modeling. Finally, it introduces an architectural model that integrates FAIR infrastructures and proteoform resolution. In addition, practical recommendations for making AI-ready proteomics, including a minimal community checklist to support reproducibility, benchmarking, and translational scalability, are provided. Full article

(This article belongs to the Section Proteomics Technology and Methodology Development)

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20 pages, 872 KB

Open AccessReview

Proteostasis, Assisted Reproductive Technologies, and Neurodevelopmental Differences: An Integrative Perspective

by Alberto Fucarino, Yousef Mohamadi, Francesco Cappello, Federica Scalia, Giulia Russo, Giuseppe Gullo and Leila Noori

Proteomes 2026, 14(2), 19; https://doi.org/10.3390/proteomes14020019 - 21 Apr 2026

Abstract

Proteostasis, defined as the coordinated regulation of protein synthesis, folding, trafficking, and degradation, is essential for maintaining cellular integrity and supporting normal development. During reproduction and early life stages, efficient proteostasis is crucial for gamete quality, successful fertilization, embryonic development, and neurodevelopmental outcomes. [...] Read more.

Proteostasis, defined as the coordinated regulation of protein synthesis, folding, trafficking, and degradation, is essential for maintaining cellular integrity and supporting normal development. During reproduction and early life stages, efficient proteostasis is crucial for gamete quality, successful fertilization, embryonic development, and neurodevelopmental outcomes. Increasing evidence suggests that impaired proteostasis contributes to infertility and may be intertwined with biological vulnerabilities associated with assisted reproductive technologies [ARTs]. This review provides an integrative perspective on the role of disrupted proteostasis in infertility, ART procedures, and neurodevelopmental differences [NDD]. We review epidemiological and molecular findings indicating proteostasis failure in both male and female infertility, with particular emphasis on molecular chaperones. Among these, heat shock protein 60 [Hsp60] is discussed as a central mediator linking mitochondrial function, protein quality control, and reproductive competence. We further highlight that ART procedures coincide with sensitive periods of epigenetic reprogramming and proteostasis regulation during early embryogenesis, indicating that disturbances in proteostasis may affect epigenetic stability and subsequent neurodevelopmental outcomes. In addition, this review emphasizes the importance of proteoforms and proteome complexity as critical determinants of reproductive success and neurodevelopmental robustness in the context of ART. Finally, we discuss the potential of proteomic and chaperone-based biomarkers as emerging tools to optimize ART strategies, improve gamete and embryo selection, and enhance risk assessment and clinical outcomes. The current review underscores proteostasis as a fundamental yet underrecognized mechanism linking reproductive biology, ART outcomes, and long-term neurodevelopment while highlighting future directions for translational investigations. Full article

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3 pages, 379 KB

Open AccessCorrection

Correction: Banu et al. The Proteome of Dictyostelium discoideum Across Its Entire Life Cycle Reveals Sharp Transitions Between Developmental Stages. Proteomes 2026, 14, 3

by Sarena Banu, P. V. Anusha, Pedro Beltran-Alvarez, Mohammed M. Idris, Katharina C. Wollenberg Valero and Francisco Rivero

Proteomes 2026, 14(2), 18; https://doi.org/10.3390/proteomes14020018 - 21 Apr 2026

Abstract

In the original publication [...] Full article

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20 pages, 4205 KB

Open AccessArticle

Computational Phosphosite-Specific Network Analysis of YES1 Y426 Reveals Cancer-Associated Phosphorylation Patterns

by Afreen Khanum, Leona Dcunha, Suhail Subair, Athira Perunelly Gopalakrishnan, Akhina Palollathil and Rajesh Raju

Proteomes 2026, 14(2), 17; https://doi.org/10.3390/proteomes14020017 - 16 Apr 2026

Abstract

Background: YES1 is an Src family non-receptor tyrosine-protein kinase that regulates cell growth, migration, survival, and oncogenic signaling. Although YES1 activation mechanisms and substrates have been extensively studied, its phosphosite-specific regulation across diverse biological contexts remains poorly understood. Methods: We performed a large-scale [...] Read more.

Background: YES1 is an Src family non-receptor tyrosine-protein kinase that regulates cell growth, migration, survival, and oncogenic signaling. Although YES1 activation mechanisms and substrates have been extensively studied, its phosphosite-specific regulation across diverse biological contexts remains poorly understood. Methods: We performed a large-scale integrative analysis of 3825 publicly available human mass spectrometry-based phosphoproteomic datasets to map YES1 phosphorylation events. Co-modulation, co-occurrence, evolutionary conservation, and disease-association analyses were conducted to characterize the functional and clinical relevance of site-specific YES1 phosphorylation. Results: Y426 emerged as the predominant YES1 phosphosite across diverse biological conditions, localized within the activation loop of the kinase domain and conserved across Src family kinases. Co-modulation analysis identified 421 positively and 102 negatively associated phosphosites enriched in biological processes related to cell cycle regulation, transcription, cytoskeletal remodeling, apoptosis, and carcinogenesis. Among these high-confidence protein phosphosites, we identified 24 binary interactors, 5 upstream regulators, and 8 candidate downstream substrates. Comparison with DisGeNet cancer biomarkers showed overlap between YES1-associated phosphoproteomic signatures and site-specific oncogenic markers across multiple cancers, such as breast cancer, colorectal cancer, leukemia, and lung adenocarcinoma. Conclusions: This study provides a systems-level, phosphosite-focused view of YES1 signaling and supports a central regulatory role for Y426 within global phosphoregulatory and cancer-associated networks. Full article

(This article belongs to the Section Multi-Omics Studies that Include Proteomics)

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29 pages, 768 KB

Open AccessReview

Beyond Reanalysis: Critical Issues in Data Reuse for Solid Tumor Proteomics

by Federica Franzetti, Nicole Giugni, Manuel Airoldi, Heather Bondi, Tiziana Alberio and Mauro Fasano

Proteomes 2026, 14(2), 16; https://doi.org/10.3390/proteomes14020016 - 7 Apr 2026

Abstract

Proteomics represents a fundamental layer for understanding the molecular complexity of solid tumors by quantifying protein abundance and capturing proteoforms and post-translational modifications undetected in genomics or transcriptomics analyses. As mass spectrometry-based technologies and public proteomics repositories have expanded, opportunities for large-scale data [...] Read more.

Proteomics represents a fundamental layer for understanding the molecular complexity of solid tumors by quantifying protein abundance and capturing proteoforms and post-translational modifications undetected in genomics or transcriptomics analyses. As mass spectrometry-based technologies and public proteomics repositories have expanded, opportunities for large-scale data reuse have grown accordingly. Nevertheless, data availability has not been translated into straightforward reuse: differences in experimental design, acquisition strategies, quantification workflows and metadata quality still limit the reproducibility and cross-study comparability. In this review, proteomics data reuse is defined as the systematic reanalysis and integration of publicly available datasets to support precision oncology applications such as biomarker assessment and antibody–drug conjugate target prioritization. We discuss reuse as an end-to-end analytical process, focusing on data analysis workflows, harmonization strategies, and the impact of heterogeneous experimental and analytical choices on interoperability. The increased application of artificial intelligence in proteomics data integration and reuse is also addressed, highlighting its analytical potential while underscoring the risks of overinterpretation when biological context and data structure are not adequately considered. Using colorectal and prostate cancer as representative examples, we illustrate how proteomics data reuse can support biological discovery and translational research, while critically examining the factors that limit robustness and clinical relevance. Full article

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20 pages, 1900 KB

Open AccessArticle

Proteomic Insights into the Immune and Sex-Specific Proteins in the Skin Mucus of Barramundi (Lates calcarifer)

by Varsha V. Balu, Dean R. Jerry and Andreas L. Lopata

Proteomes 2026, 14(1), 15; https://doi.org/10.3390/proteomes14010015 - 20 Mar 2026

Abstract

Background: Fish skin mucus contains proteins involved in diverse biological pathways, representing a valuable non-invasive diagnostic of fish health. Methods: Skin mucus from three male and three female barramundi was analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following protein extraction and S-Trap digestion. [...] Read more.

Background: Fish skin mucus contains proteins involved in diverse biological pathways, representing a valuable non-invasive diagnostic of fish health. Methods: Skin mucus from three male and three female barramundi was analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following protein extraction and S-Trap digestion. Results and Discussion: A total of 1801 protein groups were matched to the L. calcarifer reference proteome and functionally annotated using Gene Ontology (GO) terms via UniProt ID mapping, with representation across Biological Process, Cellular Component, and Molecular Function categories. Functional classification using eggNOG-mapper further associated leading protein group sequences with Clusters of Orthologous Groups (COGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. GO-based screening prioritised 352 putatively immune-relevant protein groups and 24 protein groups associated with sex- and reproduction-related processes, highlighting the functional complexity of the skin mucus proteome. Comparative analysis revealed sex-associated patterns in protein group detection and relative abundance, with differential abundance analysis identifying 244 protein groups exhibiting statistically significant differences between male and female samples. Conclusions: This study provides the first comprehensive discovery-based characterisation of the barramundi skin mucus proteome and establishes a baseline reference dataset for this aquaculture-relevant species. The findings support the utility of skin mucus proteomics for exploring immune and sex-associated molecular patterns and provide a baseline dataset for future validation studies investigating non-invasive health and reproductive monitoring. Full article

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