Platelets are pivotal circulating effector cells central to diverse physiological and pathological processes, with many of their essential functions—such as activation, adhesion, and signal transduction—being modulated by protein glycosylation. However, the inherent microheterogeneity and low ionization efficiency of glycopeptides continue to pose substantial technical barriers to achieving large-scale and precise analysis at the intact glycopeptide level, thereby limiting the in-depth exploration of platelet glycoproteome. In this study, we first evaluated three enzymatic digestion strategies—trypsin alone, Lys-C/trypsin combination, and Glu-C/trypsin combination, followed by optimization of mass spectrometry parameters for stepped-collision-energy high-energy collisional dissociation (sceHCD), ultimately establishing an efficient glycoproteomic workflow tailored to human peripheral blood platelets. Application of this workflow to platelets from healthy volunteers led to the successful identification and quantitation of 4696 intact N-glycopeptides mapping to 299 glycoproteins and 534 glycosites involving 447 distinct glycans. Extension to the O-glycoproteome further confirmed workflow generalizability, successfully identifying 2807 O-glycopeptides, 548 O-glycosites, and 340 O-glycoproteins. Exploratory functional analysis indicated that males were enriched with glycoproteins associated with coagulation and complement regulation, whereas for females pathways related to cell adhesion and viral infection response were enriched. These sex‑dimorphic glycoprotein signatures provide insights into sex‑related differences in platelet physiology.

Mass spectrometry-based targeted quantitation technologies are widely recognized for their high sensitivity, strong specificity, broad dynamic range, and excellent quantitative accuracy, and have been established as a cornerstone technique for discovery, validation, and translation research in the proteomics field. Ongoing advancements in mass spectrometry hardware and software have significantly enhanced targeted proteomics analysis, enabling its application in a wide range of scientific disciplines. However, several technical and methodological challenges remain. Real-time acquisition strategies and algorithms have been developed with increased efficiency and intelligence. These strategies, namely, employing stable isotope triggering, real-time search localization, or retention time (RT) adjustment, have transformed targeted proteomics from a static analytical model into a dynamic and intelligent acquisition framework. This transition enables the detection of more targets per run while enhancing sensitivity, specificity, and quantification accuracy. This review focuses on novel strategies and technologies in mass spectrometry-based targeted proteomics. It provides a systematic description of the underlying principles, applications, and technical challenges of these strategies and technologies, with the intention to provide a comprehensive resource for an in-depth understanding of targeted quantitative proteomics.

Pancreatic ductal adenocarcinoma (PDAC) carries a poor prognosis largely due to lack of efficient diagnostic means. We applied mass spectrometry-based high-coverage plasma proteome analysis accompanying with machine learning to develop a 5-protein diagnostic model: SNCA, GCLC, LBP, ALAD, and SORD. For differentiating PDAC from healthy controls (HCs), this model reached an area under the curve (AUC) of 0.973 with 100% sensitivity and 85% specificity in the discovery cohort, with nested cross-validation confirming robust performance (AUC = 0.958). Further validation centered on SNCA achieved an AUC of 0.835 in an independent validation cohort. SNCA also showed good diagnostic performance in PDAC patients with low CA19-9 level (AUC = 0.868), underscoring its potential value for this subgroup. Overall, these findings indicate SNCA as a promising candidate plasma diagnostic marker for PDAC.

To ensure the stability and efficacy of recombinant collagen during development, storage, and application, it is essential to establish systematic and reliable stability assessment methodologies. This study developed and validated an integrated multi-technique analytical strategy for the comprehensive evaluation of the thermal stability of recombinant humanized type III collagen (rhCol III). The strategy combines size exclusion chromatography-multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), circular dichroism spectroscopy (CD), SDS-PAGE, and µLC-MS/MS to perform correlative analyses of physical aggregation, secondary structure, chemical modifications, and biological functions in samples stored under accelerated degradation conditions (40 °C) and control conditions (4 °C) for three months. The results demonstrate that the established strategy can systematically differentiate and quantify degradation pathways under different temperature conditions. SEC-MALS, SDS-PAGE and DLS revealed that high-temperature storage significantly increased the formation of large aggregates (>100 nm), while circular dichroism spectroscopy confirmed that the secondary structure remained largely intact. The core of the strategy, µLC-MS/MS-based post-translational modification (PTM) analysis, quantitatively elucidated key chemical modifications: oxidation (up to 16.86%) and deamidation (up to 12.31%) levels were markedly elevated, whereas proline-4-hydroxylation, essential for structural stability, was substantially reduced (<6%). Key modification sites (e.g., N425, M131, and P144/P132) were successfully localized and found to be enriched in thermally sensitive regions N- and C-termini. This chemical modification profile was highly consistent with functional assay results: samples stored at 4 °C exhibited significantly superior promotion of cell adhesion (+42% vs. +21%) and migration (45.28% vs. 42.05%) compared to those stored at 40 °C. Moreover, the study found that a higher moisture content in the lyophilized samples not only significantly reduced aggregate formation but also affected the levels of PTMs. The analytical strategy developed in this study enables multidimensional and correlative assessment of protein stability. It not only provides a key stability-indicating methodology for rhCol III but also offers an analytical reference framework for stability studies, process optimization, and quality control of biomacromolecular materials.

Abstract Plasma proteomics is expanding across platforms and cohorts, and integrating these data for AI demands comparability at the protein level, not merely concordant associations 1,2 . Affinity and MS platforms use distinct probes (antibodies, aptamers, or peptides) and signal readouts, yielding contradictory cross-platform results 3,4 . Without a known quantitative truth, we cannot distinguish biology from measurement distortion, leaving no gold standard for integration. Here we introduce Plasmix—a plasma reference suite with predefined male:female ratios (M, 1:0; Y, 3:1; P, 1:1; X, 1:3; F, 0:1)—and show that preserving this quantitative titration gradient, not just technical repeatability, predicts cross-platform concordance and identifies protein measurements suitable for integration. Profiling Plasmix across five platforms (Olink, SomaScan, NULISA, AAgAtlas, and MS-DIA) and 12 protocols across 17 batches, we found discordance is dominated by signal generation, not sample identity, and platforms distort signals in a protein-specific manner. Crucially, proteins retaining the titration response showed stronger agreement in an independent cohort; anchoring to the Plasmix midpoint (P) via sample-to-reference ratios reduced distortions, extending harmonizable coverage by 10–20%. Plasmix thus provides a physical ruler to benchmark accuracy, identifying genuinely integrable measurements before pooling datasets or training AI models—a critical bottleneck for plasma proteomics.

Regional diversity may influence the composition of human milk fat globule membrane (HMFGM) protein composition and protein glycosylation. Here, we first systematically evaluated the efficiency and stability of six methods for the extraction of HMFGM proteins from breast milk. The optimal method (methanol-chloroform precipitation of MFGM proteins, followed by solubilization in 0.4% SDS lysis buffer) was further applied to the proteome and N-glycoproteome analysis of 49 breast milk samples from five regions across China. Proteomic analysis revealed the geographically divergent expression of BST2, COCH, FUCA1 and FGFBP1 in MFGM. The parallel N-glycoproteomic profiling identified 4914 site-specific N-glycans mapping to 689 glycoproteins, 699 glycosylation sites, and 420 glycan structures. This multiomics study reveals region-specific variations in the molecular composition of HMFGM proteins and their N-glycoprotein derivatives, elucidating the structural and functional dynamics of the human HMFGM in representative regions of China.

We established a streamlined ubiquitomics workflow for FFPE tissues, identifying over 10 000 GlyGly sites. With this workflow, we showed that storage duration had a minor effect on the stability of ubiquitomics information for 7 years.

Background Histidine residues are crucial for protein structure and function, playing key roles in metal coordination, catalysis, and post translational modifications. Comprehensive profiling of functional histidine sites remains technically challenging because of the insufficient chemoselectivity of labeling reagents, and the lack of robust enrichment methods compatible with complex proteomes. Methods Seven histidine-targeting probes representing three distinct reaction mechanisms were systematically evaluated using a peptide-centric workflow. Reaction conditions were optimized at the peptide level via mass spectrometry, and large-scale assessments of labeling efficiency, site selectivity, and sequence preferences were performed using tryptic digests of HeLa cell lysates. Results Nucleophilic substitution-based probes preferentially modify histidine in charged microenvironments, whereas nucleophilic addition reagents exhibit broad sequence tolerance and achieve the highest labeling coverage. In contrast, singlet oxygen-mediated probes display increased site-selectivity for C-terminal and dynamically accessible histidine sites, yielding highly confident labeling products with improved biocompatibility. Among all evaluated reagents, acrolein achieved the highest labeling efficiency, while 1-methyl-4-phenyl-1,2,4-triazoline-3,5-dione exhibited superior histidine specificity and compatibility with downstream enrichment strategies. Conclusion This work provides a comparative framework for histidine-targeted chemical proteomics, establishes optimized labeling and analysis pipelines, and highlights complementary probe reactivities that can be combined with click chemistry for comprehensive histidine profiling in complex biological systems. Nevertheless, the peptide-level study may not fully reflect the reaction in intact proteins due to steric hindrance.

Determining the drug targets and off-targets is crucial and can provide key information for the drug action mechanism. As one of the multiple methods used to identify the drug targets, thermal proteome profiling (TPP) is more direct, simpler, and suitable for large-scale applications. TPP can also be used at the live-cell level to obtain more information about the pathway regulation beyond the direct targets. At present, there are three formats of TPP: temperature range TPP (TPP-TR), compound concentration range TPP (TPP-CCR), and two-dimensional TPP (2D-TPP). TPP-TR is the most widely practiced form and has been improved in different aspects. In this review, we systematically discuss the evolution, advantages, and limitations of TPP-TR sample preparation and data analysis to provide potential strategies for the development and application of innovative methods.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism, and are typically present at low levels in therapeutic monoclonal antibody (mAb) and other recombinant biopharmaceutical products. Firstly, a high-pH–low-pH “two-dimensional” reversed-phase nano-LC–MS/MS label-free quantification (2D nano-LC–MS/MS LFQ) method with a robust stability (CV

An in-depth exploration of molecular composition of human milk could provide a scientific basis for the development of substitutes. The present study was conducted to analyze human milk proteins from 110 individuals from five regions of China and across three stages of lactation to investigate the change patterns. We developed a micro-flow liquid chromatography tandem mass spectrometry (mu LC-MS/MS) system with data- independent acquisition (DIA) proteomics technology that can rapidly and stably characterize the human milk proteome. In total, 2796 proteins were identified. Among these proteins, CPM, ACSL1, and RPL13 changed significantly during lactation, and SCP2, GALK1 and GALE changed significantly between regions. Bioinformatics analysis revealed that human milk is altered by complex interactions between genetic and environmental factors. Our results not only reveal the regional and longitudinal patterns of change in human milk proteome but also provide theoretical basis and technical support for the production and quality control of infant formula.

The concept of 'proteomics-driven precision medicine' highlighted in 2019 emphasized the potential of proteomics to transform precision medicine by offering deeper insights into dynamic biological processes. Since then, this field has seen remarkable advancements, interlinking with key pillars such as protein expression profiling, post-translational modifications, protein-protein interactions and spatial proteomics to transform healthcare. Technological progress has led to the creation of comprehensive reference maps of proteomes, identification of over 90% of human protein-coding genes, and detailed cellular and molecular landscapes within organs. Proteomics has significantly advanced health monitoring and disease surveillance by developing aging models, predicting disease risks with superior protein risk scores, identifying biomarkers for early conditions like dementia, and securing Food and Drug Administration (FDA) approval for multiple cancer biomarkers. By providing deeper insights into the proteome's complexity and dynamics, proteomics is revolutionizing our understanding, diagnosis and treatment of diseases, firmly establishing itself as a cornerstone of precision medicine.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a five-year survival rate of only 12 %. Exact diagnosis and intervention are critical for improving patient prognosis. Core fucosylation (CF) of proteins plays a vital role in the progression of various cancers, including PDAC. In this study, we analysed differences in site-specific CF glycopeptides and corresponding glycoproteins between paired tumour tissues (Ts) and normal adjacent tissues (NATs) from patients with PDAC via a specific enrichment and cleavage strategy. We identified 1447 CF glycoproteins with 2654 CF glycopeptides and revealed that CF glycoproteins are involved in various biological processes. A panel of candidate biomarkers was determined via nested cross-validation and receiver operating characteristic (ROC) analysis with bootstrap resampling and further validated in an independent cohort. This work revealed that several CF glycopeptides from Decay-accelerating factor (CD55), Versican (VCAN), Carboxypeptidase Z (CPZ)and Mucin 16 (MUC16) have the potential to distinguish PDAC NATs from Ts, with an area under the curve (AUC) of 1 in the validation cohort. These data demonstrate that CF glycoproteins may associated with PDAC development and progression and have potential as candidate biomarkers for clinical decision-making.

Capsaicin, the primary active ingredient and irritant in chili peppers, has been utilized across multiple fields as a food adductive or because of its potential anticancer, antioxidant, anti-inflammatory and metabolic regulatory properties. Despite its diverse uses, the mechanism of action of capsaicin has not been fully revealed. Here, we investigated the changes in the proteome and phosphoproteome of A549 cells upon treatment with capsaicin for different durations and at different doses. Pressure cycling technology (PCT) was applied for rapid sample preparation and digestion, significantly improving the stability of phosphorylated proteins and allowing in-depth phosphoproteome analysis within 6 h with protein inputs of 100 μg. Proteomic and phosphoproteomic alterations can be used to accurately identify perturbations caused by various capsaicin doses and exposure durations. Proteomic analysis revealed that capsaicin administration affected the cell cycle and DNA damage pathways in a time- and dose-dependent manner. Compared with the proteomic changes, more sensitive and rapid alterations were observed in the phosphoproteome, a finding further supported by posttranslational modification (PTM) set enrichment analysis (PTM-SEA) of the phosphoproteomic data. The phosphorylation status of serine protein kinase, Aurora kinase A, and Aurora kinase B changed faster than their protein expression. Overall, the findings here identify the proteomic and phosphoproteomic alterations caused by capsaicin, providing new insights for multiomics analysis to elucidate chemical perturbations.

Protein SUMOylation is a newly discovered process similar to protein ubiquitination and is crucial for protein stability and protein localization. SAE1 is an important enzyme that initiates protein SUMOylation, but its role in the progression of non-small cell lung cancer remains unknown. We analyzed the protein expression profiles of non-small cell lung cancer tissues and single-cell sequencing data and confirmed that SAE1 is highly expressed in non-small cell lung cancer cells and is associated with a malignant phenotype. Knockdown of SAE1 decreased the growth, cell cycle progression, and metastasis of non-small cell lung cancer cells both in vitro and in vivo. Mechanistically, using the protein expression profile of non-small cell lung cancer cell lines with altered SAE1 expression, we showed that SAE1, a key molecule mediating protein SUMOylation, can SUMOylate the epithelial-mesenchymal transition-related protein N-cadherin, stabilize N-cadherin and promote the occurrence of the EMT in non-small cell lung cancer cells, leading to lung cancer invasion and metastasis. In clinical application, we used sputum samples from patients with lung cancer or chronic pulmonary obstructive pulmonary disease for protein profiling and further used sputum-based thin-slice technology for experimental verification, which confirmed the application potential of SAE1 in the diagnosis of lung cancer patients. In summary, our findings reveal a critical role for SAE1 as an oncogene in lung cancer cells and suggest that SAE1 may be used for the diagnosis of lung cancer patients.

Protein phosphorylation modification is one of the key regulatory mechanisms in cellular signaling transduction and metabolic processes. The phosphorylation state of target proteins is regulated by specific protein kinases and phosphatases, which add or remove phosphate groups. Histidine phosphorylation (pHis) plays a crucial role in both prokaryotes and eukaryotes life activities and is linked to various pathological processes. Unlike the stable phosphorylation of proteins via phosphate ester bonds, histidine phosphorylation is linked through phosphoramide bonds, making it highly sensitive to high temperatures and low pH. This sensitivity has historically impeded progress in identifying and studying histidine phosphorylation. In recent years, the development of new techniques in phosphoproteomics and the emergence of pHis-specific antibodies have promoted the identification and functional research of pHis-modified substrates. For the first time, more than 700 pHis-modified proteins have been identified in mammalian cells, and pHis-modified substrates such as focal adhesion kinase (FAK) and phosphoglycerate mutase 1 (PGAM1) have been found to promote tumor development. This article mainly reviewed the key mechanisms and functions of histidine kinases and histidine phosphatases in regulating the histidine phosphorylation of specific substrates, and highlights their significant roles in human physiological and pathological processes, aiming to provide guidance for further research into the biological functions of histidine phosphorylation.

Alpha-1,6 core fucosylation (CF) is a unique glycoform of N-glycans, and studies showed that CF modifications are involved in the occurrence and progression of various diseases and may provide potential disease biomarkers. Current strategies for the CF glycoproteome are often based on multistep enrichment of glycoproteins or glycopeptides and sequential cleavage with different glycosidases to truncate the N-glycans. Although the detection ability of low-abundance glycoproteins is improved, sample loss, high cost, and the time-consuming multistep operation also affect the reproducibility of results and the practicality of the method. Here we developed a single-step truncation (SST) strategy and evaluated its potential for the CF glycoproteome of human serum. The SST strategy has the advantages of fewer operational steps, lower cost, higher number of identifications, and better quantitative stability compared with previous approaches and provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome. (c) 2024 Wiley Periodicals LLC.Basic Protocol 1: Single-step truncation strategy for core fucosylation glycoproteome analysis in human serumBasic Protocol 2: Liquid chromatography-tandem mass spectrometry quantification of site-specific core fucosylation glycopeptidesAlternate Protocol: Pretreatment of cellular samples of core fucosylation glycoproteome with single-step truncation strategy

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.

Background: Histidine phosphorylation (pHis) plays a key role in signal transduction in prokaryotes and regulates tumour initiation and progression in mammals. However, the pHis substrates and their functions are rarely known due to the lack of effective analytical strategies. Results: Herein, we provide a strategy for unbiased enrichment and assignment of the pHis peptides. First, the entire procedure was designed under alkaline conditions to maintain the stability of the N-P bond of pHis and high-pH reverse-phase chromatography was used to efficiently separate the pHis peptides. Second, exploiting the coelution benefits of diethyl labelling, the ratios of light- and heavy-labelled peptides were accurately quantified, and the sites of phosphorylated histidine were assigned. Finally, Cu-IDA bead enrichment and data-independent acquisition mass spectrometry analysis were used to improve the coverage of the histidine phosphoproteome. With this novel strategy, 768 and 1125 potential pHis peptides were identified from lysates of E. coli and HeLa cells, respectively. And these values represent the highest coverage of the histidine phosphoproteome for both cell types. Significance: These data strongly support the presumption that pHis modifications are widely present in bacteria. The study provides an efficient strategy and can lead to a better understanding of pHis-modified substrates and their biological functions.