Clinical proteomics最新文献

{"title":"Scalable clinical proteomics from DNA/RNA extraction flowthroughs enables integrated proteogenomic profiling from a single cancer biopsy.","authors":"Filip Mundt, Annelaura Bach Nielsen, Juanjuan Wang, Josephine Kerzel Duel, Christina Westmose Yde, Martina Amnitzbøll Eriksen, Ulrik Lassen, Finn Cilius Nielsen, Kristoffer Rohrberg, Matthias Mann","doi":"10.1186/s12014-026-09632-1","DOIUrl":"10.1186/s12014-026-09632-1","url":null,"abstract":"<p><strong>Background: </strong>Precision oncology has largely relied on genomic and transcriptomic sequencing to guide therapy selection in patients with advanced cancer. However, these molecular layers do not fully capture the functional state of tumors, as proteins largely represent the active effectors of cellular processes and signaling pathways. Integrating proteomic and phosphoproteomic measurements with genomic data has therefore emerged as an important objective in translational oncology. In routine clinical workflows, tumor biopsies are frequently processed using DNA/RNA extraction kits such as the Qiagen AllPrep system, which generates a protein-rich flowthrough that is typically discarded.</p><p><strong>Methods: </strong>Here we systematically evaluated whether the residual protein fraction generated during Qiagen AllPrep DNA/RNA extraction can serve as a reliable source for mass spectrometry-based proteomics in clinical tumor biopsies. Within the Copenhagen Prospective Personalized Oncology (CoPPO) precision oncology trial, residual protein fractions from metastatic cancer biopsies were recovered, precipitated, and processed using optimized workflows for liquid chromatography-mass spectrometry. We established procedures for protein recovery, digestion, and peptide cleanup, and evaluated multiple acquisition strategies including data-dependent acquisition (DDA) and data-independent acquisition (DIA). We further assessed sample stability during long-term storage, analytical reproducibility, scalability, and compatibility with phosphoproteomics.</p><p><strong>Results: </strong>Optimized single-shot workflows consistently generated deep proteomes from AllPrep-derived biopsy material, quantifying more than 10,000 protein groups in DIA analyses. Protein integrity was preserved in samples stored at - 80 °C for up to five years. The resulting proteomes retained tissue-associated biological signatures and quantified protein products corresponding to approximately 71% of genes represented on the FoundationOne^® CDx panel. Phosphoproteomic analyses identified more than 10,000 phosphorylation sites associated with key oncogenic signaling pathways including AKT1, BRAF, and EGFR. Implementation of short liquid chromatography gradients enabled high-throughput analysis of up to 60 proteomes per day without compromising data quality.</p><p><strong>Conclusions: </strong>Residual protein fractions from Qiagen AllPrep DNA/RNA extraction provide a scalable resource for deep proteomic and phosphoproteomic profiling from the same biopsy used for genomic and transcriptomic analyses. This workflow enables integrated proteogenomic characterization from a single clinical sample and provides a framework for integrated proteogenomic studies in precision oncology.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":"23 1","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13616122/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149058818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated proteomics and machine learning for identifying candidate serum biomarkers in acute myocardial infarction-complicated cardiogenic shock: a prospective exploratory study.","authors":"Xi Wang, Qian-Feng Xiao, Fang-Yang Huang, Si Wang, Ying Xu, Xiao-Bo Pu, Yan Yang, Mao Chen, Xin Wei","doi":"10.1186/s12014-026-09626-z","DOIUrl":"https://doi.org/10.1186/s12014-026-09626-z","url":null,"abstract":"<p><strong>Background: </strong>Cardiogenic shock secondary to acute myocardial infarction (AMI-CS) prohibitively impacts survival. This prospective study aimed to discover and internally verify candidate serum protein biomarkers and evaluate their potential prognostic value for 30-day mortality in AMI-CS patients.</p><p><strong>Methods: </strong>AMI-CS patients were consecutively enrolled into discovery (n = 30) and verification (n = 60) cohorts. Candidate biomarkers were screened using Data-Independent Acquisition (DIA) mass spectrometry, analyzed via differential abundance and weighted gene co-expression network analysis (WGCNA), and verified via targeted Parallel Reaction Monitoring (PRM). Boruta feature selection for five machine learning algorithms were embedded within a rigorous nested cross-validation scheme. Incremental prognostic value over clinical predictors was evaluated using Cox regression and metrics including the integrated discrimination improvement (IDI).</p><p><strong>Results: </strong>DIA proteomics identified 216 proteins differentially abundant between 30-day survivors and non-survivors, and WGCNA defined an outcome-associated module linked to shock severity and enriched for oxidative stress and energy metabolism. During PRM verification, leakage-free nested cross-validation random forest model selected a seven-protein panel (YWHAZ, QDPR, MDH2, FAH, PSMA1, FABP5, and AHCY), which achieved a mean area under the ROC curve of 0.82 (95% CI: 0.69-0.95) for 30-day mortality. Incorporating this panel significantly enhanced the IDI of the SCAI model (IDI 0.143, P = 0.016) and the IABP-SHOCK II model (0.138, P = 0.034), which remained independent after adjusting for core clinical confounders.</p><p><strong>Conclusion: </strong>This exploratory study identifies a seven-protein candidate panel with potential value for early short-term risk stratification in AMI-CS patients. Large-scale multi-center external validation is strictly warranted to confirm its real-world clinical utility.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148535503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted proteomics for the detection of androgen receptor variants in preclinical models of castration-resistant prostate cancer.","authors":"Zoi E Sychev, Gabrianne Larson, Megan L Ludwig, Seiei Shiba, Kiel Tietz, Joshua W Russo, Steven Balk, Eva Corey, Stephen Plymate, Emmanuel S Antonarakis, Scott M Dehm, Jesse C Seegmiller, Justin M Drake","doi":"10.1186/s12014-026-09623-2","DOIUrl":"10.1186/s12014-026-09623-2","url":null,"abstract":"<p><strong>Background: </strong>The emergence of Androgen Receptor variants (AR-Vs) during hormonal treatment is believed to be one of the key mechanisms of resistance. AR-Vs lack the ligand-binding domain and function as constitutively active transcription factors, rendering AR-targeted endocrine-based therapeutics ineffective in tumors that express AR-Vs. The goal of this study was to develop a targeted multiplex assay to identify known and unexplored AR-V proteins and help guide the stratification of PCa patients based on their AR-V protein profiles.</p><p><strong>Methods: </strong>A targeted multiple reaction monitoring (MRM) assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to quantify total AR proteins using the AR N-terminal domain (AR-Nterm), a well-characterized AR splice variant AR-V7 (AR-V7), a putative, previously identified but uncharacterized protein isoform (AR-V12) and isoforms that code for an exon 3 duplicated junction seen in AR-V2 and AR- full length with two copies of exon 3. Specimens included three AR-positive PCa cell lines (22Rv1, VCaP16, LNCaP), two AR-negative PCa cell lines (PC3, DU145), and a patient-derived xenograft (PDX) model of CRPC. Endogenous target peptides were quantified according to tier-2 targeted mass spectrometry \"fit-for-purpose\" guidelines to ensure analytical rigor.</p><p><strong>Results: </strong>Analytical performance was characterized across a linearity range of 4.15 fmol to 4000 fmol followed by evaluations of precision, accuracy, selectivity, stability, ion suppression and repeatability studies. The overall coefficient of variation (CV) was < 20% across seven independent validation experiments. Endogenous AR-V peptides were accurately quantified using calibration curves, achieving acceptable linear regression coefficients (R² > 0.95). Four targets, including AR-Nterm and 3 AR-variants, were identified and quantified in PCa cell lines and in a PDX pre-clinical model.</p><p><strong>Conclusion: </strong>This study establishes a robust, validated MRM targeted proteomics assay capable of reproducibly detecting and quantifying both known and previously uncharacterized AR-Vs at the protein level  . The assay demonstrated < 20% analytical variation at the lower limit of quantification of 4.25 femtomole (fmol) for AR-Nterm and 8.25 fmol for AR-V7, AR Exon3-Dup/AR-V2 and AR-V12 and, showed robust analytical performance in preclinical models and is ready for further evaluation in clinical specimens. This provides a clinically applicable platform for the comprehensive profiling of AR-V proteins in human subjects, enabling improved stratification of patients with CRPC and supporting precision therapeutic strategies.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13670591/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148429951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inverse co-variation of circulating PIWIL2 and PIWIL4 in gastric adenocarcinoma: an exploratory ELISA-based study.","authors":"Mariam Abuladze, Elene Kekelia, Ian Alexander Cree, Eter Dumbadze, Ekaterina Kldiashvili","doi":"10.1186/s12014-026-09621-4","DOIUrl":"10.1186/s12014-026-09621-4","url":null,"abstract":"&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Gastric cancer is characterized by substantial molecular heterogeneity, and RNA-mediated regulatory mechanisms may contribute to its biological complexity. PIWI-family proteins are central components of the PIWI-interacting RNA pathway, but their circulating patterns in gastric adenocarcinoma remain insufficiently characterized. This study aimed to describe circulating PIWIL1, PIWIL2, and PIWIL4 protein concentrations and explore intra-cohort co-variation patterns in patients with gastric adenocarcinoma without evaluating disease specificity, diagnostic performance, or clinical utility.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;In this cross-sectional, targeted ELISA-based exploratory study, circulating PIWIL1, PIWIL2, and PIWIL4 concentrations were quantified in 93 de-identified gastric adenocarcinoma samples. PIWIL1 and PIWIL2 measurements were available for 88 samples, and PIWIL4 measurements were available for 72 samples. Commercial ELISA kits were used according to the manufacturer's instructions, but independent in-house serum validation, including dilution linearity, serum parallelism, matrix-interference testing, intra- and inter-assay precision, and orthogonal protein confirmation, was not performed. Spearman's rank correlation analysis with false discovery rate correction was used to assess exploratory intra-cohort relationships among PIWI-family proteins.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;PIWIL1 and PIWIL2 showed relatively stable circulating distributions, whereas PIWIL4 demonstrated pronounced heterogeneity and right-skewness. The most prominent rank-based relationship was an inverse correlation between PIWIL2 and PIWIL4 (Spearman's ρ ≈ -0.53, q &lt; 0.001), based on 72 complete cases. This correlation remained statistically significant in sensitivity analyses using log-transformed data and exclusion of extreme values. No statistically significant correlations were observed between PIWI-family proteins and CEA or CA 19 - 9 after false discovery rate correction. Exploratory TP53 analyses were not central to the primary objective and are reported descriptively; PIWIL2 showed a modest positive correlation with TP53 (Spearman's ρ = 0.33), while PIWIL4 showed a weak positive correlation (ρ = 0.19).&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions: &lt;/strong&gt;This study identified an inverse rank-based co-variation between circulating PIWIL2 and PIWIL4 concentrations within a de-identified gastric adenocarcinoma cohort. However, this observation should be interpreted strictly as a pre-validation, exploratory ELISA-derived intra-cohort signal. Because the study lacked comparator groups, clinical annotation, tissue-level confirmation, functional assays, orthogonal protein quantification, and independent serum validation of the ELISA measurements, the observed correlation cannot be considered an analytically validated proteomic association. Further studies with confirmed assay linearity, serum parallelism, matrix-interference assessment, between-plat","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13628752/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148390020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic and glycosylation biomarkers in rheumatoid arthritis: advancing early diagnosis and precision therapy.","authors":"Preeti Sharma, Shahbaz Aman, Priya Koundal, Shakeel Ahmed Mohammed, Hardeep Kaur, Faraz Ahmad, Sabiha Khatoon, Shafiul Haque, Arif Hussain","doi":"10.1186/s12014-026-09618-z","DOIUrl":"10.1186/s12014-026-09618-z","url":null,"abstract":"<p><strong>Background: </strong>Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint inflammation and damage. Early clinical diagnosis is crucial for effective intervention but presents significant challenges due to an initial asymptomatic inflammatory phase.</p><p><strong>Main body: </strong>Anti-citrullinated-protein antibodies (ACPA) and rheumatoid factor (RF) detection have been useful in early RA diagnosis, but their reliability is debatable as they are often non-specific. This has prompted a quest for alternative, more robust biomarkers. Researchers have turned to advanced proteomic and glycosylation analyses of biological fluids (e.g., synovial fluid, plasma and serum) and tissues using techniques such as MALDI-MS, Q-TOF, and SELDI-TOF. These approaches may offer a comprehensive approach to scrutinize a range of biomolecules as potential RA biomarkers, from citrullinated-proteins and peptides to novel potential protein biomarkers such as thymosin, macrophage-capping protein, calgranulins, and serum amyloid-A. Further, proteomics-based approaches have the ability to specifically monitor changes in the RA proteome (e.g., glycosylated VCAM1/SEMA4D proteins and GFAP/A1BG auto-antibodies) to unearth potential diagnostic and prognostic candidates. Disease monitoring in response to anti-rheumatic drugs using treatment-responsive markers, such as S100A8/A9 heterocomplex and leucine-rich alpha-2 glycoprotein, is another application of proteomics-based technologies.</p><p><strong>Conclusion: </strong>In view of the significance of prediction, early diagnosis and monitoring of RA symptomatology, this review discusses the potential utilities of proteinaceous species as proteomics-based biomarkers that may provide insights into disease mechanisms and offer potential avenues for personalized therapeutic interventions to revolutionize RA management.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13629044/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148390034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive biochemical insights into derivatives of PSA and glycosylation-specific changes in PSA: role in diagnosis and management of prostate cancer.","authors":"Laxmidhar Das","doi":"10.1186/s12014-026-09617-0","DOIUrl":"10.1186/s12014-026-09617-0","url":null,"abstract":"<p><strong>Background: </strong>Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide, and prostate-specific antigen (PSA) test is currently the standard of choice for PCa diagnosis. Globally, several methods are employed for diagnosis of PCa, and each holds a unique role and importance. Serum PSA test and Magnetic Resonance Imaging (MRI) are widely used for diagnosis of PCa; however, MRI has certain limitations and disadvantages compared to PSA test. Moreover, growing evidences suggest that solely simple testing of serum PSA levels to diagnose PCa leads to widespread overdiagnosis and overtreatment. This is because simple PSA test has certain limitations, including lack of specificity, elevation in Benign Prostatic Hyperplasia (BPH) and inability to detect a significant number of PSA-negative tumors. Additionally, serum PSA levels do not directly correlate with higher grades and stages of PCa.</p><p><strong>Main body: </strong>The purpose of this manuscript is to provide comprehensive and up-to-date knowledge, critical perspectives, and practical applications of derivatives of PSA and glycosylation-specific changes in PSA, with the aim of minimizing unnecessary and excessive biopsy in patients suspected of having PCa. Therefore, the use of various derivatives of PSA was focused here to increase the sensitivity and specificity of serum PSA for diagnosis of PCa. In addition to PSA levels, glycan structure of PSA changes with progression of PCa, which suggest that aberrant PSA glycosylation increases as PCa progresses. Differences in the glycan structure of PSA enable to distinguish normal PSA from cancerous origin, suggesting an important biochemical application of glycan structure of PSA in the diagnosis of PCa. Thus, present study highlights the role of derivatives of PSA and glycosylation-specific changes in PSA in the diagnosis and management of PCa. Additionally, this article emphasizes integration of glycan-based assays with PSA derivatives into multi-analyte panels, paving the way for more sensitive and specific diagnosis of PCa and aiding in the management of PCa.</p><p><strong>Conclusion: </strong>The manuscript will provide extensive information to minimize superfluous biopsies, identify patients truly needing biopsy, differentiate BPH from PCa, distinguish indolent from aggressive PCa, enable early diagnosis and treatment at early-stage, monitor treatment response and early detection of recurrence.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13621508/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148344112","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic analysis of malignant ascites and its impact on ovarian cancer spheroids.","authors":"Jack Scanlan, Parul Mittal, Jennifer Washington, Clifford Young, Michael Antoniou, Marina Kochetkova, Martin K Oehler, Peter Hoffmann, Manuela Klingler-Hoffmann","doi":"10.1186/s12014-026-09619-y","DOIUrl":"10.1186/s12014-026-09619-y","url":null,"abstract":"<p><strong>Background: </strong>Most advanced ovarian cancer patients develop malignant ascites, which describes a buildup of fluid in the peritoneal cavity caused by increased vascular permeability and obstructed lymphatic drainage. Malignant ascites contains cancer cells, which can aggregate as spheroids, as well as stromal cells, cancer-associated fibroblasts, and blood cells that create a complex tumor microenvironment. This study explores the proteome of ascites and how this environment affects the viability, phenotypes, proteomes, and treatment responses of ovarian cancer cells.</p><p><strong>Methods: </strong>Using label-free proteomics, we compared the proteomes of cell-free malignant ascites from ovarian cancer patients with those of serum. Additionally, we examined the ex vivo chemotherapy responses of cancer spheroids cultured in ascites. Through detailed proteomic analysis of cells grown as 2D or 3D in tissue culture medium or ascites, we identified biological pathways and specific proteins induced by ascites. Finally, we performed orthogonal validation of a candidate marker, TGM2, using immunofluorescent staining.</p><p><strong>Results: </strong>Proteomics of cell-free ascites identified increased levels of extracellular, secreted, and membrane proteins when compared to serum. Ascites enhanced the baseline cell viability and spheroid formation of immortalized ovarian cancer cell lines compared to standard cell culture medium. However, chemotherapy-induced cell death of spheroids grown in standard cell culture medium remained proportional to changes observed in ascites-cultured spheroids. Ascites-driven phenotypic changes were not recapitulated by adding selected chemokines nor periostin to the cell culture medium, suggesting that additional factors are required. Notably, ascites induced similar ECM, secreted, and membrane proteins across 2D and 3D models, including TGM2, which was validated in spheroids through immunofluorescent staining.</p><p><strong>Conclusions: </strong>This study contributes to our understanding of the role of ascites in the regulation of the proteome and viability of cancer cells. It provides evidence for the induction of TGM2 expression by ascites. Results from this pilot study warrant further study in a larger cohort.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13621715/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148344076","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Repurposing the liquid-based Pap test for the detection of ovarian cancer protein biomarkers.","authors":"Amy P N Skubitz, Kristin L M Boylan, Limeng Liu, Ashley Petersen, Yuqian Gao, Joshua R Hansen, Jesse B Trejo, Thomas L Fillmore, Yang Wang, Melissa A Geller, Peter A Argenta, Samantha Hoffman, Karin Rodland, Tao Liu, Paul D Piehowski","doi":"10.1186/s12014-026-09610-7","DOIUrl":"10.1186/s12014-026-09610-7","url":null,"abstract":"<p><strong>Background: </strong>Earlier detection is strongly associated with increased survival for women with ovarian cancer. Unfortunately, current screening strategies, employing serial serum or ultrasound assessments, lack adequate sensitivity and specificity for use in the low-prevalence general population. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. Since ovarian cancer cells have been observed in Pap tests, ovarian cancer protein biomarkers may also be present; yet Pap samples have not been rigorously examined for diagnostic proteins. Assessment of cervical effluent, as can be collected in a Pap test, has the potential to differentiate ovarian cancer cases from healthy controls, and thereby demonstrate that intra-abdominal pathology may be detected using this commonly acquired specimen. We hypothesize that proteins shed by ovarian cancer cells can be detected in the SurePath™ liquid-based Pap test fixative using mass spectrometry (MS)-based proteomics, making it possible to distinguish women with ovarian cancer from healthy women.</p><p><strong>Methods: </strong>Candidate ovarian cancer biomarkers were successfully identified in liquid-based Pap test samples from 20 cases of high grade serous ovarian cancer, 10 benign ovarian conditions, and 10 healthy control samples, by performing Tandem Mass Tag™ isobaric labeling, 2D liquid chromatography-MS/MS, and bioinformatics integration. Selected reaction monitoring (SRM) MS-based targeted proteomics was then performed using a panel of candidate biomarkers to quantify their abundance in an expanded patient cohort of 90 liquid-based Pap tests.</p><p><strong>Results: </strong>A multi-protein classifier was developed using the SRM-MS data comprised of 6 proteins and achieving an AUC of 0.880 (95% CI: 0.738-0.989); sensitivity at 90% specificity was 0.800.</p><p><strong>Conclusion: </strong>This pilot study provides evidence that the cervical effluent collected in SurePath™ fixative from Pap tests has the potential to be used as a biospecimen for the detection of ovarian cancer proteins. Our long-term goal is to develop a noninvasive screening test that can be incorporated into a routine Pap test or a self-administered home test, so that women can be screened simultaneously for cervical and ovarian cancer.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13282856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148270631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pairing of high-mannose glycans in human IgE-Fc: implications for secretion and stability.","authors":"Sakurako Nomura, Tatsuro Iwabuchi, Haruka Sawataishi, Kizashi Kamo, Noriyoshi Manabe, Ryohei Uematsu, Izumi Sakamoto, Kenji Hirose, Eunsang Kwon, Yoshiki Yamaguchi","doi":"10.1186/s12014-026-09616-1","DOIUrl":"10.1186/s12014-026-09616-1","url":null,"abstract":"<p><strong>Background: </strong>Immunoglobulin E (IgE) is the least abundant antibody class in serum, but plays a central role in type I allergic responses. The Fc region of human IgE (IgE-Fc) contains four potential N-glycosylation sites: Asn265 and Asn371 are modified with complex-type glycans; Asn394 predominantly carries a high-mannose glycan; and Asn383 remains unmodified. Despite the recognized importance of glycosylation in antibody function, the structure-function relationships of the individual IgE-Fc glycans remain poorly understood. This study aimed to elucidate the structural and functional significance of N-glycans on IgE-Fc, particularly the high-mannose glycan attached to Asn394.</p><p><strong>Methods: </strong>The expression and secretion of recombinant human IgE-Fc constructs including wild-type IgE-Fc, a high-mannose-deficient mutant (N394Q), and a triple mutant lacking complex-type glycans (N265Q/N371Q/N383Q) was assessed in mammalian Expi293F cells. In addition, the thermal stability of wild-type IgE-Fc treated with Endo H or PNGase F was evaluated by a thermal shift assay. Glycan compositions and pairing patterns in IgE-Fc were characterized by intact mass spectrometry (MS) and liquid chromatography-MS analysis of released glycans.</p><p><strong>Results: </strong>Wild-type IgE-Fc and the triple mutant were stably expressed, whereas secretion of the high-mannose-deficient mutant (N394Q) was markedly impaired, demonstrating that the Asn394 glycan is essential for proper folding and efficient secretion. Endo H treatment significantly decreased the melting temperature of wild-type IgE-Fc, indicating that high-mannose glycans contribute to structural stability. Integration of MS data on the intact protein and LC-MS data on the enzyme-released N-glycans revealed that high-mannose glycans at Asn394 associate in a non-selective manner between heavy chains.</p><p><strong>Conclusions: </strong>These findings establish that the high-mannose glycan at Asn394 is required for both the secretion and structural stability of IgE-Fc. Furthermore, the observation of non-selective glycan pairing between heavy chains provides insight into the temporal coordination of disulfide bond formation and N-glycan processing during IgE biosynthesis, thereby advancing our understanding of the molecular mechanisms underlying IgE structure and function.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13523533/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148257618","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative mass spectrometry analysis of high and low centrifugation extracellular vesicle (EV) pellets from healthy urine following Tamm-Horsfall protein removal.","authors":"Dana Bielopolski, Luca Musante, Henrik Molina, Douglas Barrows, Samantha Upson, Lifang Yang, Thomas Carrol, Jonathan N Tobin, Rhonda G Kost, U Erdbrügger","doi":"10.1186/s12014-026-09615-2","DOIUrl":"10.1186/s12014-026-09615-2","url":null,"abstract":"<p><strong>Objectives: </strong>Urinary extracellular vesicles (uEVs) are potential biomarkers for renal physiology and disease. Their isolation is challenged by Tamm-Horsfall protein (THP) contamination, particularly in high-speed ultracentrifugation (P100) fractions. Low-speed (P20) fractions contain biologically relevant uEVs, yet their proteomic composition is underexplored. This study evaluates strategies for THP removal to optimize mass spectrometry analysis and characterizes P20 and P100 uEV proteomes and its combination in healthy urine.</p><p><strong>Methods: </strong>Urine from a healthy volunteer was processed via sequential centrifugation at 21,200 g (P20) and 164,244 g (P100). THP removal was performed using low ionic strength buffer (P20) and size-exclusion chromatography (P100). Western blotting, nanoparticle tracking, and mass spectrometry were used to characterize protein content. Functional analysis was performed to assess biological significance.</p><p><strong>Results: </strong>Mass spectrometry identified 1,103 unique proteins in P100, 929 in P20, and 1,676 in the combined pellet, with 90% similarity between fractions. P20 was enriched in proteasome components (PSMC2, PSMD2, PSMD5, PSMD6, PSMD8, PSMD11, PSMD13, PSME1), while P100 had no advantage in terms of expression profile in any of the proteins examined. Functional analysis indicated immune activation in P100 and protein degradation in P20, suggesting complementary biological roles.</p><p><strong>Conclusion: </strong>This study reveals proteasome enrichment in P20 uEVs and supports a combined P20 + P100 approach to improve biomarker discovery. Optimized THP removal strategies enhance mass spectrometry resolution, facilitating uEV analysis for clinical and research applications. INSTITUTIONAL REVIEW BOARD FOR HEALTH SCIENCES RESEARCH (IRB-HSR) AT UVA: HSR220053.</p>","PeriodicalId":10468,"journal":{"name":"Clinical proteomics","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2026-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13523286/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148249440","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}