Proteomes

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

29 pages, 3062 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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14 pages, 8721 KB

Open AccessReview

Emergence of Catalytic Activity in VRK3: Phosphoproteomic Insights into the Regulatory Network of a Former Pseudokinase

by Ayadathil Sujina, Amal Fahma, Suhail Subair, Rajesh Raju and Poornima Ramesh

Proteomes 2026, 14(1), 14; https://doi.org/10.3390/proteomes14010014 - 18 Mar 2026

Abstract

Vaccinia-Related Kinase 3 (VRK3) is increasingly recognized as a crucial signaling modulator in both normal and pathological processes. This kinase was long thought of as a catalytically inactive pseudokinase, until recently it was established to phosphorylate Barrier to Autointegration Factor (BAF) proteins through [...] Read more.

Vaccinia-Related Kinase 3 (VRK3) is increasingly recognized as a crucial signaling modulator in both normal and pathological processes. This kinase was long thought of as a catalytically inactive pseudokinase, until recently it was established to phosphorylate Barrier to Autointegration Factor (BAF) proteins through its extracatalytic domain. VRK3 regulates diverse cellular pathways through scaffold interactions and context-dependent phosphorylation. This review is centered around the phosphoregulatory network that modulates VRK3 phosphorylation with implications in its abundance and function. A large-scale phosphoproteomic data integration was performed by combining phosphoproteomics profiling and differential phosphorylation from 115 mass spectrometry studies, identifying 32 high-confidence phosphorylation sites on VRK3. Notably, VRK3 (S59), (S82), and (S83) were predominantly observed highlighting plausible functional significance. These phosphorylation sites share 33 potential upstream kinases, and multiple interactor proteins, which in combination are known to regulate ERK, Hippo, and GPCR pathways. These insights advance the understanding of phosphorylation control by kinases and highlight opportunities to target VRK3-associated networks for therapeutic intervention in diseases such as glioma and liver cancer. Full article

(This article belongs to the Section Proteome Bioinformatics)

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15 pages, 1617 KB

Open AccessArticle

Dimethyl Sulfoxide Enhances HLA Peptide Identification

by Terry C. C. Lim Kam Sian, Yue Ding, Scott A. Blundell, Ralf B. Schittenhelm and Pouya Faridi

Proteomes 2026, 14(1), 13; https://doi.org/10.3390/proteomes14010013 - 13 Mar 2026

Abstract

Background: Mass spectrometry (MS)-based immunopeptidomics has emerged as the gold standard for profiling HLA-bound peptides, yet detection remains challenging due to their non-tryptic nature, variable lengths, and lack of basic residues, which limit ionisation and fragmentation efficiency. Methods: To address these limitations, we [...] Read more.

Background: Mass spectrometry (MS)-based immunopeptidomics has emerged as the gold standard for profiling HLA-bound peptides, yet detection remains challenging due to their non-tryptic nature, variable lengths, and lack of basic residues, which limit ionisation and fragmentation efficiency. Methods: To address these limitations, we investigated the impact of incorporating 5% dimethyl sulfoxide (DMSO) into LC-MS/MS mobile-phase buffers on immunopeptidomic workflows. Using B-lymphoblastoid cell lines expressing HLA class I and II alleles and elastase-digested HeLa lysates as a surrogate for non-tryptic peptides, we assessed peptide identification, ionisation efficiency, charge state distribution, and fragmentation quality. Results: DMSO significantly increased peptide identifications across all sample types, with gains of ~1.33 folds for HLA class I, ~1.55 folds for HLA class II, and ~1.24 folds for elastase digests. Improvements were systematic and reproducible, driven by enhanced electrospray ionisation, higher charge states, and superior MS2 spectral quality, evidenced by ~2-fold increase in b- and y-ion intensities. Importantly, DMSO did not introduce major sequence bias, preserving motif integrity and predicted binding characteristics. Conclusions: Overall, these findings establish DMSO as a robust additive for improving sensitivity and reliability in immunopeptidomics, particularly for low-input or clinically derived samples. Full article

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

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15 pages, 510 KB

Open AccessReview

Proteomic Analysis in Search of New Biomarkers of Immune Thrombocytopenia (ITP)—A Review of Current Data

by Anastasia Boura-Theodorou, Konstantina Psatha, Stefania Maniatsi, Areti Kourti, Georgia Kaiafa, Michalis Aivaliotis and Kali Makedou

Proteomes 2026, 14(1), 12; https://doi.org/10.3390/proteomes14010012 - 12 Mar 2026

Abstract

Immune thrombocytopenia (ITP) is a hematological disorder commonly found in individuals of any gender, race, or age. Patients with ITP will present with thrombocytopenia either in a primary form or because of an infection or a dysfunction in the immune system. The severity [...] Read more.

Immune thrombocytopenia (ITP) is a hematological disorder commonly found in individuals of any gender, race, or age. Patients with ITP will present with thrombocytopenia either in a primary form or because of an infection or a dysfunction in the immune system. The severity of ITP is linked to diminished production of platelets due to the blockage of production in the bone marrow niche and increased destruction of platelets, which confirms the diagnosis of the disorder. The investigation of the pathogenesis of ITP is of critical importance as it can give an important indication of the state of the patient, guiding us through risk assessment and treatment. Proteomics can provide tools to explore the protein profile of ITP. In this review, we aimed to uncover different biomarkers, both diagnostic and prognostic, that have been investigated with proteomic methodologies and that might help in understanding the pathogenesis of ITP and providing personalized treatment to patients. Several differentially abundant proteins were identified, including haptoglobin isoforms, heat shock proteins (HSPA6, HSPA8), integrin β3 (ITGB3), 14-3-3 protein eta (YWHAH), vitamin D-binding protein, fibrinogen chains, MYH9, and FETUB, which are involved in key signaling pathways, such as PI3K/akt, TNF-a, and mTOR, and they demonstrate potential as diagnostic and prognostic biomarkers. Collectively, current data support the value of proteomics for uncovering the molecular landscape of ITP and guiding the development of precision diagnostics and personalized therapeutic strategies. Full article

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34 pages, 14889 KB

Open AccessArticle

Cellular Responses of Maize Roots to Long-Term Cadmium Exposure: Adjustments of Class III Peroxidases, Plasma Membrane and Tonoplast Sub-Proteomes

by Sabine Lüthje, Ayse Gül Yilmaz, Kalaivani Ramanathan, Waldemar Gräfenstein, Jenny M. Tabbert, Stefanie Wienkoop, Katrin Heino, François Clement Perrineau and Sönke Harder

Proteomes 2026, 14(1), 11; https://doi.org/10.3390/proteomes14010011 - 25 Feb 2026

Abstract

Background: Crop plants have to deal with long-term cadmium exposure to farmlands contaminated by intensive use of fertilizers and pesticides. For uptake and sequestration, Cd²⁺ has to pass the plasma membrane and tonoplast. Class III peroxidases, plasma membrane, and tonoplast sub-proteomes were [...] Read more.

Background: Crop plants have to deal with long-term cadmium exposure to farmlands contaminated by intensive use of fertilizers and pesticides. For uptake and sequestration, Cd²⁺ has to pass the plasma membrane and tonoplast. Class III peroxidases, plasma membrane, and tonoplast sub-proteomes were studied. Methods: Control and Cd²⁺-treated maize (Zea mays L.) were grown in hydroponics for 18 days. Soluble peroxidases were partially purified by chromatofocusing and characterized by substrate specificity. Membrane-bound peroxidases were analyzed spectrophotometrically and by non-reducing SDS-PAGE. Soluble and plasma membrane-bound peroxidases were identified by mass spectrometry. Shotgun proteomics was used to identify membrane proteins of differential abundance. Results: Guaiacol peroxidase activities increased in soluble fractions of Cd²⁺ samples. A Cd²⁺-specific soluble peroxidase (ZmPrx101) was identified, and ZmPrx85 abundance increased significantly in the plasma membrane. Substrate specificity of peroxidases revealed a preference for ferulic acid and esculetin, which was confirmed by docking analyses. Primary active transporters increased auxin efflux (brachytic2, ABCB9, and ABCB21), Cd²⁺ exclusion (ABCG34), and sequestration into the vacuole (HMA2, ABCB27). Evaluation of sub-proteome fractions demonstrated significant changes for proteins involved in disease resistance responses and cell wall modification. Conclusions: Molecular adjustments of maize root proteome to long-term Cd²⁺ exposure revealed relevance of low-abundant proteins for Cd²⁺ tolerance and putative stress markers. Full article

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

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14 pages, 986 KB

Open AccessArticle

Comparison of the Trapping Efficiency for Tryptic Peptides on Particle-Packed and Micro-Pillar Trap Columns for Proteomics Analyses

by Jadranka Miletić Vukajlović, Bojana Ilić, Bella Bruszel, Tanja Panić-Janković and Goran Mitulović

Proteomes 2026, 14(1), 10; https://doi.org/10.3390/proteomes14010010 - 18 Feb 2026

Abstract

Background: Low-volume trapping columns are essential for sample enrichment, desalting, and injection profile focusing on nano-LC–MS-based proteomics. They enable higher sample loading, improve chromatographic performance, and protect the analytical column by removing salts and contaminants. Recently, monolithic trap columns with micropillar architecture have [...] Read more.

Background: Low-volume trapping columns are essential for sample enrichment, desalting, and injection profile focusing on nano-LC–MS-based proteomics. They enable higher sample loading, improve chromatographic performance, and protect the analytical column by removing salts and contaminants. Recently, monolithic trap columns with micropillar architecture have emerged as alternatives to conventionally packed traps. This study compares the performance of a packed and a micropillar monolithic trap column for the analysis of tryptic peptides. Methods: A tryptic digest of HeLa cell lysate was analyzed under identical LC–MS conditions using both trap types. Peptides were detected at 214 nm and analyzed by nano-ESI on a Q Exactive Plus Orbitrap. Data were searched against the human UniProt database (February 2023) using FragPipe v20.0, and statistical evaluation of MaxLFQ intensities was performed in Perseus using Welch’s t-test and clustering analysis. Results: Over 2500 proteins were identified with both setups. The packed trap column yielded more total peptides, particularly those with post-translational modifications and higher hydrophilicity, whereas the monolithic column favored peptides of intermediate hydrophobicity. Chromatographic profiles confirmed a slight reduction in the trapping efficiency of hydrophilic peptides by the monolithic trap. Conclusions: Trap column design significantly influences peptide recovery and proteome coverage. Full article

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

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20 pages, 2949 KB

Open AccessArticle

Scout-Triggered Multiple Reaction Monitoring Enables Robust Quantification of Host Cell Proteins Across Bioprocess Matrices

by Julie Flecheux, Chloé Bardet, Laura Herment, Tanguy Fortin and Jérôme Lemoine

Proteomes 2026, 14(1), 9; https://doi.org/10.3390/proteomes14010009 - 17 Feb 2026

Abstract

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to [...] Read more.

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices. Methods: Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented. Results: This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances. Conclusion: Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis. Full article

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

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26 pages, 2509 KB

Open AccessArticle

Proteome-Wide Analysis of Functional Phosphosites in the FGFR Family of Proteins: Insights from Large-Scale Phosphoproteomic Analysis

by Akhina Palollathil, Althaf Mahin, Athira Perunelly Gopalakrishnan, Tejaswini R Poojari, Alimath Sambreena, Prathik Basthikoppa Shivamurthy and Rajesh Raju

Proteomes 2026, 14(1), 8; https://doi.org/10.3390/proteomes14010008 - 13 Feb 2026

Abstract

Background: Fibroblast growth factor receptors (FGFRs) play a crucial role in tissue homeostasis and organ development by regulating cellular processes, including proliferation, differentiation, and survival. Dysregulation of FGFRs contributes to developmental disorders and carcinogenesis. As membrane-bound receptors, they represent promising targets for therapeutic [...] Read more.

Background: Fibroblast growth factor receptors (FGFRs) play a crucial role in tissue homeostasis and organ development by regulating cellular processes, including proliferation, differentiation, and survival. Dysregulation of FGFRs contributes to developmental disorders and carcinogenesis. As membrane-bound receptors, they represent promising targets for therapeutic intervention and drug development. Methods: This study employed a systematic in silico analysis of publicly available phosphoproteomics datasets to provide a comprehensive overview of the phosphorylation regulatory network of the FGFR family. Results: We identified predominant phosphosites in FGFR1-4 that exhibited differential abundance across diverse experimental conditions, specifically, Y653 in FGFR1; S453, Y586, Y656, and Y657 in FGFR2; S444 and S445 in FGFR3; and S573 in FGFR4. Our analysis identified 32 and 89 significantly co-modulated phosphosites on other proteins with FGFR3 and FGFR4, respectively. Beyond the upstream kinases from the FGFR family, we also identified MAPK1 as a potential upstream kinase of FGFR4. Furthermore, disease enrichment analysis revealed that proteins co-modulated with FGFR3 were primarily involved in skeletal developmental disorders, such as brachydactyly, short toe, and syndactyly of fingers, whereas those associated with FGFR4 were linked to various cancers. Conclusions: Our findings highlight key disease-associated phosphosites within the FGFRs and offer a foundation for advancing phosphosite-focused therapeutic research. Full article

(This article belongs to the Section Proteome Bioinformatics)

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19 pages, 2370 KB

Open AccessArticle

PTMs_Closed_Search: Multiple Post-Translational Modification Closed Search Using Reduced Search Space and Transferred FDR

by Yury Yu. Strogov, Sergey A. Spirin, Mark V. Ivanov, Maria A. Kulebyakina, Anastasia Yu. Efimenko and Oleg I. Klychnikov

Proteomes 2026, 14(1), 7; https://doi.org/10.3390/proteomes14010007 - 2 Feb 2026

Abstract

Background: Currently, post-translational modification (PTM) search in MS/MS data is performed using either open modification search (OMS) or closed search (CS) algorithms. The OMS method allows for the determination of many PTMs and unknown mass-shifts in one run. In contrast, closed search [...] Read more.

Background: Currently, post-translational modification (PTM) search in MS/MS data is performed using either open modification search (OMS) or closed search (CS) algorithms. The OMS method allows for the determination of many PTMs and unknown mass-shifts in one run. In contrast, closed search algorithms are more sensitive but limited in the number of PTMs that can be specified in one search. Methods: In this manuscript, we propose an optimized Python algorithm based on the IdentiPy search engine that performs an automated sequential search for each PTM based on previous annotations from public databases and customized protein lists. We also determined the sufficient size of the search space to increase the significance of false discovery rate (FDR) estimation. We modified the FDR calculation algorithm by implementing a spline approximation of the ratio of the modified decoys, and by calculating error propagation to filter out unstable data and determine the cutoff value. Results: The results of this pipeline for a test dataset were comparable to previously published data in terms of the number of unmodified peptides and proteins. Additionally, we identified 13 different types of peptide PTMs and achieved an increase in relative protein coverage. Our filtration method based on spline transferred FDR showed a superior number of identified peptides compared to separate FDR. Conclusions: Our developed pipeline can be used as a standalone application or as a module of multiple PTM search in data analysis platforms. Full article

(This article belongs to the Section Proteome Bioinformatics)

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Open AccessArticle

Evaluating the Impact of Two Different Diets on the Protein Profile of the Brain, Liver, and Intestine of the Barramundi

by Mohadeseh Montazeri Shatouri, Igor Pirozzi, Pinar Demir Soker, Zeshan Ali, Ardeshir Amirkhani and Paul A. Haynes

Proteomes 2026, 14(1), 6; https://doi.org/10.3390/proteomes14010006 - 29 Jan 2026

Abstract

Background: Commercial feed formulations are increasingly being evaluated for their nutritional impacts on aquaculture species, yet the molecular consequences of commonly used commercial diets remain underexplored. Methods: This study investigated the effects of two commercial diets, diet A (higher land animal protein) and [...] Read more.

Background: Commercial feed formulations are increasingly being evaluated for their nutritional impacts on aquaculture species, yet the molecular consequences of commonly used commercial diets remain underexplored. Methods: This study investigated the effects of two commercial diets, diet A (higher land animal protein) and diet B (higher fish meal content), on the protein profile in the brain, liver, and intestine of barramundi (Lates calcarifer). A 12-week feeding trial was conducted with controlled water quality, and proteomic profiling was performed using data-independent acquisition. Results: Differential analysis revealed consistent changes between diets across all tissues, with a higher percentage of differentially abundant proteins observed in between-diet comparisons (12.99% in brain, 12.73% in liver, and 16.59% in intestine) than within-diet controls (<8%), confirming a measurable dietary effect size. In total, 3901 proteins in the brain, 3660 in the liver, and 5025 in the intestine were quantified. Functional enrichment highlighted upregulation of ferroptosis pathways, downregulation of apelin signaling in the brain, and increased digestive proteases in the liver. ICP-MS confirmed elevated iron concentrations in the brain, liver, and intestine of fish fed on diet B. Conclusions: These findings demonstrate that molecular pathways linked to iron metabolism, digestion, and growth regulation are very sensitive to dietary composition, highlighting how proteomics can help identify subtle impacts of compositional differences in aquaculture feeding. Although physiological parameters did not differ significantly, the proteomic alterations observed across tissues likely indicate organ-specific metabolic adaptations to the differing nutrient availability between diets. Full article

(This article belongs to the Section Animal Proteomics)

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23 pages, 8773 KB

Open AccessArticle

Integrated Phosphoproteomics Identifies TGFβ-Dependent Phosphorylation Events Linking Kinase Signaling to Autophagy in Palatogenesis

by Xia Peng, Jing Chen, Xiaoyu Zheng, Xige Zhao, Yijia Wang, Xiaotong Wang and Juan Du

Proteomes 2026, 14(1), 5; https://doi.org/10.3390/proteomes14010005 - 23 Jan 2026

Abstract

Background: Cleft palate (CP) is a prevalent craniofacial malformation, with the TGFβ pathway playing a critical role. Recent evidence links autophagy to regulating mouse embryonic palatal mesenchyme (MEPM) cells, but its interaction with TGFβ-activated phosphorylation cascades remains largely unknown. Here, we investigated the [...] Read more.

Background: Cleft palate (CP) is a prevalent craniofacial malformation, with the TGFβ pathway playing a critical role. Recent evidence links autophagy to regulating mouse embryonic palatal mesenchyme (MEPM) cells, but its interaction with TGFβ-activated phosphorylation cascades remains largely unknown. Here, we investigated the interplay between these pathways during palatogenesis. Methods: H&E and IHC analyses revealed increased expression of Beclin 1 and LC3 during the critical period of palatal shelf elevation and fusion (E13.5–E15.5). Bulk RNA sequencing (Bulk RNA-seq) further revealed enrichment of autophagy-related pathways and their interaction with TGFβ signaling. TMT-based phosphoproteomics was performed on TGFβ2-treated MEPM cells. Results: We identified 23,471 peptides and 3952 proteins, including 6339 phosphopeptides corresponding to 2195 phosphoproteins. Differential analysis found 477 phosphopeptides with increased abundance and 53 with decreased abundance, revealing the enrichment of seven serine (p-Ser) motifs (RxxS, SDxD, SDxE, SP, SxDE, SxEE, SxxxxD) and one threonine (p-Thr) motif (TP). Notably, kinase-substrate enrichment analysis identified CSNK2A as a previously unrecognized phosphorylation regulator, together with MAPKs and CDKs. Functional enrichment showed significant involvement of mTOR, MAPK, and autophagy/mitophagy pathways. Conclusions: Our findings revealed that TGFβ2 reshapes the MEPM phosphoproteome through Smad-independent pathway, expanding the palate-specific phospho-signaling atlas beyond the canonical Smad cascade. Full article

(This article belongs to the Section Animal Proteomics)

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