The venom of the South American rattlesnake Crotalus durissus terrificus (Cdt) is renowned for its minimalist yet highly toxic arsenal. Intraspecific variability in expressed proteoforms and toxin content has been previously observed in Cdt venom, resulting in heterogeneous biological activities. In light of this, our study conducted an in-depth mass spectrometry-based analysis, combined with structural prediction to comprehensively assess Cdt venom variability and sexual dimorphism. Using peptidomic analysis, we identified 76 native peptides within Cdt venom, predominantly (63%) associated with noncanonical processing of crotoxin subunits, and 9 novel peptides via de novo sequencing. Quantitative analysis showed increased abundance of two crotapotin-derived peptides in females, while three novel peptides were significantly increased in males. Through quantitative proteomics, we characterized a repertoire of 33 proteins, with over one-third displaying sex-based differences in abundance. Furthermore, variant analysis revealed 12 novel toxin variants, generally at lower abundance than previously described proteoforms. Predictive three-dimensional structure modeling revealed conserved scaffolds, suggesting preservation of their biological activities. Addressing venom variability provides valuable insights into the intricate variability of Cdt venom, contributing to the understanding of its biological, medical, and biotechnological implications.
Nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) underpins modern quantitative proteomics, yet the column-to-mass spectrometer interface remains an important yet often underappreciated determinant of analytical depth, sensitivity, and reproducibility. Here, we benchmark an integrated workflow comprising the newly developed OptiSpray ion source and a micropillar array column (μPAC) cartridge against a conventional Nanospray Flex Source with an Accucore resin-packed capillary column. We performed a TMTpro 18-plex experiment across nine human cell lines on a FAIMS Pro-equipped Orbitrap Exploris 480. Following basic-pH reversed-phase fractionation, 12 fractions were analyzed on both workflow configurations under matched chromatographic gradient and acquisition conditions. Across both configurations, we quantified >9000 protein groups with highly comparable quantitative reproducibility and principal component clustering. Direct comparison of protein abundance ratios across cell lines showed agreement (Pearson R2 ≈ 0.7-0.8) without systematic bias. These results were achieved without workflow-specific optimization of the OptiSpray-μPAC platform, enabling direct transfer of established acquisition methods. Despite differences in column architecture, both configurations delivered comparable proteome coverage and quantitative fidelity. These findings establish the OptiSpray-μPAC workflow as a standardized alternative to conventional capillary-based interfaces, offering simplified operation while preserving quantitative performance.
Glioblastoma (GBM) is the most common primary brain cancer in adults and remains fatal, with a median survival of a few months. There is an urgent need to develop novel therapeutic strategies against this aggressive malignancy. Modern cancer research increasingly focuses on personalized therapies tailored toward unique molecular features of each tumor or patient. In this context, cell surface proteins (CSPs) represent an attractive class of therapeutic targets due to their accessibility and central roles in physiological and pathological processes, making them among the most targeted proteins in current drug development. In this study, promising CSPs were identified through an untargeted proteomics approach using high-resolution mass spectrometry on patient-derived GBM stem-like cell (GSC) cultures, complemented by RNA-seq data and computational database analyses. From this primary discovery, five CSPs, namely PTK7, PTPRZ1, OSMR, CSPG4, and IGDCC4, were selected for detailed investigation. A targeted UHPLC-multiple reaction monitoring (MRM) method was developed and optimized to assess their expression and evaluate their abundance variations across different GSC cultures and cell passage levels. Beyond confirming these CSPs as potential therapeutic targets in GBM, our study demonstrates the value of three-dimensional GSC cultures as robust models for biomarker research and target assessment.
Alterations in circulating bile acids (BAs) have been reported in inflammatory bowel disease (IBD), but the consistency of these changes across clinically relevant comparisons remains unclear. Our goal was to investigate systemic BA alterations in IBD using a metabolomics meta-analysis with an exploratory analysis of BA-related gene expression as a supporting context. A systematic review and meta-analysis of 28 metabolomics studies examined blood BA profiles associated with IBD, IBD diagnosis, and disease activity assessment. Univariate analysis and logistic regression modeling of two independent IBD cohorts explored the blood BA-related genes and IBD. Across 28 studies that comprised 5056 IBD patients, 1721 healthy controls, and 314 non-IBD patients, 131 BAs were reported. Eight predefined clinical comparisons were eligible for the meta-analysis. Lower secondary BA levels were consistently observed in IBD patients compared with controls, between UC and CD, and in active versus remission patients. Deoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid were frequently decreased, whereas glycocholic acid was increased in certain comparisons. Transcriptomics analyses revealed differential expression of several BA-related genes in blood, including SLC51A, ABCB4, and ACOT8, across the comparisons. Our findings identify consistent circulating BA alterations in IBD and highlight the relevance of blood BA for future biomarker research in the diagnosis and disease activity assessment.
As a class of immune checkpoint inhibitors (ICIs), programmed cell death protein-1 (PD-1) blockade has demonstrated remarkable efficacy in the treatment of various malignancies. However, their clinical application is constrained by the high incidence of immune-related adverse events (irAEs), which arise from nonspecific immune activation and can affect multiple organ systems, with severe cases posing life-threatening risks. This study integrated high-throughput proteomic and metabolomic analyses to systematically characterize the molecular features associated with irAEs in cancer patients receiving PD-1 inhibitor therapy. The results showed that, following the first treatment, patients who developed irAEs exhibited potential involvement of the NF-κB pathway, along with lower baseline levels of SNRPA and higher expression of CD63. Metabolomic analyses further revealed that the kynurenine/tryptophan ratio was significantly elevated in the irAE group both at baseline and post-treatment compared with patients who did not develop irAEs. In addition, significant differences in the abundance of specific lipids were observed between the two groups prior to the administration of immunotherapy. Our findings provide exploratory insights into immune and metabolic alterations associated with PD-1 blockade treatment and may help generate hypotheses for future studies on early irAE risk assessment in cancer patients undergoing PD-1 blockade therapy.
In Brazil, approximately 25,000 snakebites occur annually, with Bothrops atrox responsible for most cases. Local morbidity is high, driven primarily by snake venom metalloproteases (SVMPs). The major SVMPs in B. atrox venom, Atroxlysin-Ia (ATXL) and Batroxrhagin (BATX), efficiently hydrolyze extracellular matrix proteins, inducing rapid hemorrhage and dermonecrosis. Thus, we characterized the composition of the exudate produced after SVMPs injection into the mice gastrocnemius muscle using proteomics. Muscle damage was evaluated by histological analysis. The composition of the exudate was analyzed by mass spectrometry. The SVMPs induced disorganization of muscle fibers and inflammatory cell migration. However, ATXL-induced a significantly higher neutrophil influx compared to BATX, likely triggered by an increase in CXCL16, suggesting a superior inflammatory capacity. In summary, despite being metalloproteases, these toxins exhibit distinct pathological profiles: ATXL is predominantly inflammatory, while BATX is more hemorrhagic. Interestingly, while endogenous serine proteinase levels were similar in both exudates, BATX showed significantly higher levels of proteinase inhibitors. Furthermore, identification of peptide bond cleavage sites revealed a pattern consistent with trypsin-like serine proteinases. These findings suggest that SVMPs not only damage tissue directly but also associate with the activation of host endogenous proteinases, which may contribute to the complex pathology of B. atrox envenomation, although direct causation remains to be established.
Snake venom variation has important clinical implications, yet individual-level venomics remains limited. We investigated inter- and intrapopulation variability in forest-steppe adder Vipera nikolskii and its recognition by commercial V. berus antivenom using proteomic and immunological approaches. Venoms from 12 individual V. nikolskii specimens representing two geographically distinct populations (BG and KM), together with three pooled V. nikolskii and one pooled V. berus samples were analyzed by LC-MS/MS, ELISA, Western blot, and pull-down assays. Multivariate analysis revealed relative homogeneity in BG and pronounced heterogeneity in KM venoms. Area-based proteomics revealed V. berus venom enrichment in PLA2 (34.6%), SVMP (14.6%), and CRiSP (15.6%), whereas V. nikolskii venoms were more variable. Pooled V. nikolskii venoms showed SVMP abundance (35.2-41.1%), contrasting with lower levels in individual samples. Antivenom binding was stronger for V. berus but weaker and more variable across individual and pooled V. nikolskii samples. Antivenom targeted PLA2/VEGF, CRiSP (only in V. berus), and Kunitz-type proteins. In vivo neutralization assay demonstrated strong protection against V. berus but not V. nikolskii venom. These findings reveal substantial compositional and antigenic variability in V. nikolskii venoms, highlight discrepancies between pooled and individual ones, and underscore the need for region-specific and functionally validated antivenom evaluation.
Abstract Antigen interference from residual antigen peptides is a persistent challenge in antipeptide immunocapture liquid chromatography–tandem mass spectrometry (IC-LC–MS/MS) assays, particularly when quantifying low-abundance protein biomarkers. Traditionally, carrier protein-conjugated surrogate peptides identical to those used in LC–MS/MS quantification are employed as antigens for immunization, often resulting in varying degrees of antigen interference from the resulting antipeptide polyclonal antibodies (pAbs). While precapture purification and other mitigation strategies can reduce interference, they may be insufficient for ultrasensitive antipeptide IC-LC–MS/MS assays. Here, for the first time, we demonstrate the use of carrier-protein-conjugated, stable isotopically labeled surrogate peptides as antigens for immunization. This strategy ensures that residual antigen peptide is distinguishable from endogenous analytes by mass spectrometry, effectively eliminating antigen interference. This paper details the design principles for stable isotopically labeled surrogate peptides used in antigen preparation and compares the performance of pAbs generated using labeled versus traditional nonlabeled peptide antigens. Results from over 20 batches of antipeptide pAbs demonstrate that this method enables the routine generation of antigen interference-free antipeptide pAbs, facilitating the development of ultrasensitive antipeptide IC-LC–MS/MS assays for low-abundance protein biomarker quantification without incurring additional time and significant cost. Data are available via ProteomeXchange with an identifier of PXD076272.
Abstract Blood-based proteomics may complement existing MCED approaches by capturing tumor-secreted proteins and systemic host responses. We reviewed prospective and multicancer studies published from 2020 to 2025 that used Olink, SomaScan, or mass spectrometry and compared evidence from case–control and prospective designs. Candidate proteins were classified as established or novel and evaluated using pathway and cancer-hallmark enrichment analyses, GEPIA3.0 Cox analyses, TCGA Kaplan–Meier survival curves, and TPCPA/RPPA protein abundance data. Across platforms, established markers, including CEACAM5, WFDC2/HE4, and GDF15 were complemented by novel candidates converging on a matrix–immune–secretory axis. This axis included MMP12 and ADAM8, CD74 and CXCL13, TGFB1, and CDCP1. Secretory and matrix-associated candidates were linked to poorer survival in several epithelial cancers, whereas immune-associated proteins showed context-dependent effects. Tumor-type-specific protein elevation supported their detectability, and interaction analysis organized the candidates into biologically coherent modules. These findings support an MCED framework combining higher-risk secretory and matrix markers with immune-context indicators to improve sensitivity and biological interpretation. Prospective validation and down-selection into scalable targeted assays are required before population-level application.
Abstract Prey rely on chemical cues to detect predators, especially in coastal ecosystems where danger is concealed by turbid waters. Juvenile oysters (Crassostrea virginica) respond to blue crab (Callinectus sapidus) chemical cues by growing stronger shells, while mud crabs (Panopeus herbstii) reduce foraging to avoid detection. However, the chemical composition of marine fear cues is largely unknown, partly due to methodological challenges. In this study, metabolomics was used to pinpoint predator metabolites that correlated with induced juvenile oyster defenses when exposed to the urine of blue crabs fed either oysters or mud crabs and to determine the chemical composition of these fear cues. Genetic algorithms applied to urine mass spectra coupled with bioassay data confirmed that metabolites from blue crab urine are responsible for fear responses by oysters. Sixteen compounds (of over 700) were determined to be potentially important cues used by juvenile oysters to detect blue crabs, with 3-(1-methyl-1H-imidazol-4-yl) propanoic acid identified as the most important contributor to this effect. These results indicate that oysters rely on certain metabolites that are ubiquitous in a variety of chemical blends to evaluate predation risk, and the response of juvenile oysters is not dependent on a singular metabolite but on numerous metabolites working in conjunction.
Abstract Helicobacter pylori (H. pylori) induces gastric precancerous lesions (GPL), but their underlying biomarkers remain poorly characterized. To establish a GPL model, BALB/c mice were inoculated with H. pylori via gavage. Immunofluorescence confirmed successful modeling by detecting H. pylori distribution and aberrant expression of precancerous lesion markers. Serum-based label-free proteomics and bioinformatic analyses were performed to screen key proteins. Data are available via ProteomeXchange (PXD076808). Western blot and immunofluorescence were used to verify key proteins and markers of DNA damage, fibrosis, and epithelial mesenchymal transition (EMT). The results revealed high H. pylori load in gastric mucosa with abnormal precancerous markers. Proteomic analysis identified 103 differentially expressed proteins and five core proteins as potential biomarkers, all overexpressed in gastric cancer. Col1a1, a fibrosis marker, was identified as a key upregulated protein and validated by Western blot and immunofluorescence. Col1a1 colocalized with the DNA damage marker γ-H2AX in gastric mucosa. H. pylori infection triggered DNA damage, fibrosis, and EMT in GPL. In conclusion, H. pylori induced DNA damage, fibrosis, and EMT in gastric mucosa, accelerating precancerous lesion progression, with Col1a1 playing a significant role.
Abstract Metabolic reprogramming fuels cancer progression, but whether common metabolic patterns exist across diverse malignancies remains incompletely understood. To address this, we integrated large-scale proteomic, transcriptomic [The Cancer Genome Atlas (TCGA)], and spatial transcriptomic (Spatial Meta-Transcriptome Database) data sets comprising 3226 samples across 24 human cancer types. Utilizing a highly controlled, predominantly patient-matched design to minimize background noise, we characterized the pan-cancer metabolic landscape via reaction-level functional task scoring, pathway enrichment, multiomics integration, network modeling, survival analysis, and microenvironmental coupling. Consistently upregulated glycan biosynthesis pathways─particularly fucosylation and sialylation─and broadly enhanced nucleotide metabolism highlighted conserved programs potentially associated with tumor growth, adaptation, and immune evasion. These signatures proved robust against variations in tumor purity across the TCGA cohorts. Furthermore, gene coexpression network modeling prioritized specific hub genes associated with this pan-cancer restructuring. Exploratory high-resolution mapping aligned tumor immunogenicity with specific glycan pathways. Notably, tumor-intrinsic glycan upregulation negatively correlates with their immune score alignment, suggesting that tumor hyperactivation may obscure immune signals in bulk tissue. Together, our results define a robust computational landscape of shared metabolic alterations, providing a data-driven framework to guide future experimental validation of candidate therapeutic targets across diverse oncological contexts.
Abstract Late-onset depression (LOD) is a common subtype of depression in the elderly, characterized by high disability and recurrence rates, along with poorly understood biological correlates, posing a serious threat to the health of the aging population; moreover, the molecular correlates and diagnostic targets for LOD remain largely unknown. Aging and stress are recognized as two core risk factors for LOD; however, whether they interact and how such interaction is associated with the depressive phenotype remain unclear, so we established a rat model of LOD to analyze the interactive effects of aging and stress on depressive-like behaviors, and combined this with cerebrospinal fluid proteomics to explore potential protein correlates and provide experimental evidence for identifying candidate therapeutic targets. In terms of results, aging and stress exhibited a synergistic association with depressive-like behaviors in LOD rats, and enrichment was observed in extracellular matrix and sphingolipid metabolic pathways; furthermore, several proteins─including ceramide, MMP2, TIMP2, SPARC, FBN1, and collagen─were identified as candidate correlates worthy of further investigation. In conclusion, stress and aging exert interactive effects on behavioral phenotypes in LOD rats, with associated alterations in extracellular matrix and sphingolipid pathways, and key molecules including Cer, MMP2, Timp2, and Sparc warrant further investigation.
Abstract Amino acid (AA) substitutions are pivotal modulators of cellular signaling and homeostasis. Current studies of AA substitutions rely mainly on genomic and transcriptomic evidence, while proteome-level characterization remains underexplored, even though such substitutions can arise beyond DNA and RNA mutations. Here we address this gap with PIPI-C, an open-search mass spectrometry tool, and establish an integrated AA substitutomics pipeline to profile AA substitutions across five cancer cohorts and dissect the regulatory roles of substitution-bearing proteins. By cross-referencing 13,399 identified AA substitutions with matched whole-exome sequencing data, we observed no overlap with patient somatic mutations. Specifically, 61% of these substitutions required two or more base alterations and could not be derived from single-nucleotide variants, rendering them exclusively detectable at the proteomic level. The remaining 39% of substitutions were compatible with single-base variations. PIPI-C captured these candidate substitutions at the proteome scale, consistent with a translational-stage origin as reported for alternate RNA decoding in mammals. We further identified notable substitutions, including F43S and E91D in hemoglobin subunit beta, P584T in filamin A, and A175N in fructose-bisphosphate aldolase B, and our pipeline provides a framework for generating hypotheses about AA substitutions in cancer escape.
Cross-linking mass spectrometry (XL-MS) has advanced as a powerful approach to map protein-protein interactions in cells. With recent advances in cross-linkers, instrumentation, and software, interactions can be observed in a proteome-wide fashion at residue-level resolution. In most XL-MS experiments, a chemical cross-linker possessing two electrophiles is added to a biological sample and records spatial information by forming covalent bonds between two proximal nucleophilic residues. On the other hand, photo-cross-linking amino acids that can be incorporated into proteins via ribosomal synthesis possess the capacity to rapidly capture transient protein-protein interactions under physiological conditions without introducing exogenous reactive species. Diazirine-based photoamino acids have been widely used for this purpose due to their small size and broad reactivity. Recent studies have shown that diazirines can form MS-cleavable linkages when they react with acidic residues. However, confident interpretation of the resulting spectra and localization of cross-linking sites remain challenging, limiting broader application of diazirine-based approaches in proteome-wide cross-linking analyses. Here, we address this limitation by leveraging DizPK, a diazirine-containing lysine analog with a dedicated MS-cleavable urea functionality. We demonstrate proteome-wide incorporation of DizPK in Escherichia coli via stochastic orthogonal recoding of translation and subsequent identification of photo-cross-linked peptides to map protein-protein interactions under physiological conditions. We find that the resulting approach can provide high-resolution structural information associated with transient biological processes.
Plaque heterogeneity underlies the propensity of atherosclerotic lesions to rupture and trigger cardiovascular events. Most proteomic studies examine bulk changes, obscuring key spatial differences in protein abundance. We report a high-resolution spatial proteomics workflow exploring the molecular landscape of human plaques and a murine myocardium. By combining laser capture microdissection with high-sensitivity ion-mobility mass spectrometry, spatial profiling of cellular and extracellular matrix (ECM) proteomes was achieved. Over 2700 proteins were detected from 50,000 μm2 areas, revealing substantial intraplaque heterogeneity across distinct regions (lipid-rich, media, shoulder, necrotic core, intima) and distance from the artery lumen. Inverse correlations between proteases (cathepsin B) and core structural ECM proteins (perlecan, HSPG2) indicated active ECM remodeling. Analysis of media layers indicated distinct protein signatures associated with smooth muscle contraction and cell-cell communication. Blood coagulation signatures, including platelet degranulation and fibrin formation, were enriched at the intima. Inflammatory (clusters of differentiation 4/68, CD4/CD68; vascular cell adhesion molecule 1, VCAM1) and vascular damage markers (tenascin-C, TNC) were enriched in shoulder regions. The necrotic core was dominated by blood proteins, consistent with intraplaque hemorrhage. This workflow resolves proteomic changes over ∼200 μm distances, providing unprecedented insights into plaque morphology and offers a powerful tool for elucidating plaque biology.
Traumatic brain injury (TBI) triggers complex neuroinflammatory cascades that involve sustained immune activation and dysregulated antibody effector functions. Immunoglobulin G (IgG) Fc N-glycosylation, particularly core fucosylation, critically modulates immune signaling through altered Fcγ receptor (FcγR) interactions; however, its role in TBI remains unexplored. Here, we developed a high-sensitivity, mass spectrometry-based glycoproteomics method for the systematic analysis of IgG Fc core fucosylation dynamics following TBI. The approach integrates Fc-specific enzymatic truncation with GlycINATOR (EndoS2) and tryptic digestion, followed by high-resolution LC-MS/MS profiling, enabling confident identification of truncated Fc glycopeptides. Furthermore, a targeted parallel reaction monitoring (PRM) strategy allowed direct quantification of core fucosylated and afucosylated glycopeptides from 10 μg of crude serum protein, eliminating the need for IgG purification. Our results reveal time-dependent and subclass-specific remodeling of IgG Fc fucosylation postinjury, characterized by an overall reduction in fucosylated species and a relative increase in afucosylation. Collectively, this study establishes a scalable analytical platform for Fc-specific glycosylation profiling and identifies IgG core fucosylation as a candidate molecular indicator of immune dysregulation in TBI, providing new insights into post-traumatic immune regulation.
The liver is a metabolically active organ vital for carbohydrate, protein, and lipid metabolism as well as detoxification of harmful substances. Here, we integrated multiple reaction monitoring (MRM) profiling, untargeted lipidomics, and desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) to identify age-associated changes in lipid profiles in the liver of adult mice across three age groups: adult (3-4 months), midaged (10 months), and old (19-21 months). Comparative lipidomic analysis revealed age-dependent remodeling of membrane phospholipids and sphingolipids. Aging was associated with significant alterations of phosphatidylcholines (PC), lysophosphatidylcholines (LPC), phosphatidylserines (PS), phosphatidylethanolamines (PE), and phosphatidylinositols (PI), characterized by reduced levels of unsaturated PC species (PC 36:5, PC 36:4) and saturated PC 32:0 alongside increased LPCs and highly unsaturated PC 40:7. In parallel, sphingolipids were also altered, with increased ceramides (Cer 34:1; O2, CerP 36:1; O2, ACer 59:1; O2) and sphingomyelins (SM 34:1; O2, SM 34:0; O2), and decreased levels of longer-chain sphingomyelins (SM 40:1; O2, SM 42:1; O2, SM 40:2; O2) compared with adult and midaged mice. Additionally, DESI-MSI revealed age-associated changes in monounsaturated (palmitoleic, oleic, eicosenoic) and polyunsaturated (linoleic, docosahexaenoic) fatty acids. These lipid alterations were consistently observed across multiple mass spectrometry platforms, including spatially resolved lipid distributions obtained by DESI-MSI, and are linked to membrane structure, signaling, and metabolism, providing new insights into age-related liver dysfunction.
Heat shock protein 90's (Hsp90) roles in cancer have prompted the development of small-molecule inhibitors that target its N-terminal ATP-binding pocket. Moreover, numerous alternative Hsp90 inhibitors (AHI) have been developed to target other sites of the Hsp90 chaperone complex. Alternative Hsp90 inhibition is typically validated in living cells by assaying the inhibitor's impact on a small number of select Hsp90 client proteins. Here, we expand these traditional characterizations by orders of magnitude, measuring changes in the proteomes of Jurkat cells treated with 10 different AHI compounds (ailanthone, celastrol, clorobiocin, coumermycin A1, daurisoline, derrubone, gambogic acid, garcinol, β-lapachone, and α-mangostin). By comparing these changes to those induced by the N-terminal Hsp90 inhibitor NVP-AUY922, we find that none of our 10 AHI compounds induce the canonical changes characteristic of N-terminal Hsp90 inhibition. Instead, six compounds appear to antagonize mitochondrial processes, while the other four demonstrate individual mechanistic profiles. We conclude that none of these 10 AHI compounds induce apoptosis in Jurkat cells via Hsp90 inhibition. Because these compounds have been previously advanced as Hsp90 inhibitors, results here indicate that insular client depletion assays are inadequate to the task of validating Hsp90 inhibition in living cells.
Profiling of newly synthesized proteins (NSPs) provides access to dynamic changes in protein production that accompany acute cellular responses. Bioorthogonal noncanonical amino acid tagging (BONCAT)-based approaches enable selective labeling of NSPs; however, their broader application remains constrained by labor-intensive enrichment workflows and limited sensitivity for direct peptide-level analysis. Here, we developed a workflow termed "Phos-tag Click Tip" by integrating a phosphorylated variant of bicyclononyne (pBCN) with Phos-tag affinity purification to selectively capture azidohomoalanine (AHA)-labeled peptides for newly synthesized proteome analysis (NSProteomics). This approach overcomes key limitations of conventional proteomics and BONCAT-based strategies by enabling efficient enrichment and sensitive detection of NSP-derived peptides. Using this workflow, we performed comprehensive NSP profiling of host cells during influenza A virus infection. We identified dynamic changes in distinct NSP profiles associated with viral replication, host restriction, and immune responses, many of which were not readily detected with conventional whole-cell- or phospho-proteomic analyses. Overall, the Phos-tag Click Tip workflow provides a complementary approach for stimulus-responsive NSP profiling, offering functionally relevant insights into host-virus interactions and cellular response mechanisms.
