Ema Svetlicic, Leonarda A. Alarcon, Roger Karlsson, Carsten Jers and Ivan Mijakovic*,
{"title":"Mass Spectrometry-Based Analysis of Surface Proteins in Staphylococcus aureus Clinical Strains: Identification of Promising k-mer Targets for Diagnostics","authors":"Ema Svetlicic, Leonarda A. Alarcon, Roger Karlsson, Carsten Jers and Ivan Mijakovic*, ","doi":"10.1021/acs.jproteome.5c00321","DOIUrl":"10.1021/acs.jproteome.5c00321","url":null,"abstract":"<p >Surface proteins of Gram-positive bacteria are critical for adherence to host tissues, evasion of the immune system, and interaction with the environment. They can be utilized as biomarkers in diagnostics, for vaccine development, and as therapeutic targets due to their accessibility and role in pathogenicity. If utilized as diagnostic targets, surface biomarkers should be highly conserved across different strains of the pathogen, unique to the species to avoid cross-reactivity, abundantly expressed on the bacterial surface, and accessible to antibodies or detection reagents. Mass spectrometry-based proteomics methods have advanced the studies of surface proteins, often in combination with selective enrichment strategies such as tryptic “shaving”. In this study, 11 clinical strains of <i>Staphylococcus aureus</i> underwent tryptic shaving to identify common surface proteins. Further bioinformatics analysis confirmed that these proteins are encoded in the core genome of <i>S. aureus</i> strains and contain species-specific peptides. In silico analysis identified 26 k-mer peptides in 15 surface proteins with structural accessibility to detection agents, making them the ideal targets for molecular diagnostics or as linear epitope targets for vaccine development or therapeutics. Among the identified candidates were known virulence-associated proteins such as PbpA, Sbi, and Asp23─previously studied in the context of vaccines─as well as uncharacterized proteins encoded by the gene loci SAUSA300_1904 and SAUSA300_1685, whose unique and surface-exposed features suggest unexplored diagnostic potential.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4575–4585"},"PeriodicalIF":3.6,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5c00321","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144792959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Edward R. Cruz, Alex N. T. Johnson, Vyas Pujari, Qi Zhang, Thao Nguyen, Michael Stadlmeier, Jessica Wang, Cristina C. Jacob, Graeme C. McAlister, Philip M. Remes* and Martin Wühr*,
{"title":"Expanding Targeted Instrumentation for Discovery Applications: Complement Reporter Ion Quantification with a Quadrupole–Ion Trap Instrument","authors":"Edward R. Cruz, Alex N. T. Johnson, Vyas Pujari, Qi Zhang, Thao Nguyen, Michael Stadlmeier, Jessica Wang, Cristina C. Jacob, Graeme C. McAlister, Philip M. Remes* and Martin Wühr*, ","doi":"10.1021/acs.jproteome.5c00356","DOIUrl":"10.1021/acs.jproteome.5c00356","url":null,"abstract":"<p >Proteomic workflows have traditionally been divided into discovery-based and targeted approaches with instrumentation optimized specifically for each. Discovery experiments typically utilize high-resolution analyzers, while targeted workflows rely on the sensitivity and specificity of triple quadrupole systems. Recently, a quadrupole–ion trap instrument (Stellar MS) has demonstrated superior performance for targeted applications compared to conventional triple quadrupoles. In this study, we expand the capabilities of this platform to multiplexed shotgun proteomics using complement reporter ion quantification in an ion trap (iTMTproC). Benchmarking experiments with defined standards show that iTMTproC achieves quantification accuracy and interference reduction comparable to MultiNotch MS<sup>3</sup> on the Orbitrap Fusion Lumos, a dedicated quadrupole–ion trap–Orbitrap tribrid instrument optimized for this purpose. Notably, iTMTproC quantifies slightly more proteins than does MultiNotch MS<sup>3</sup>. We further validate this approach through a developmental time-series analysis of frog embryos, obtaining proteomic data nearly indistinguishable from those from MultiNotch MS<sup>3</sup>, with slightly increased protein quantification depth. These findings significantly extend the functionality of targeted instrumentation, underscoring the versatility of quadrupole–ion trap systems and providing cost-effective access to highly accurate, multiplexed quantitative shotgun proteomics.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4611–4622"},"PeriodicalIF":3.6,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144787828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jing Dai*, Li Lou, Xingyao Wang, Yilian Huang, Jiao Lei, Feitai Tang, Yangyang Bian, Yong Zeng, Guangxiu Lu, Ge Lin and Shen Zhang*,
{"title":"Quantitative Phosphoproteomic Analysis of Testes from Iqcn-Deficient Mice Highlights the Significance of Calmodulin Signaling in Spermiogenesis","authors":"Jing Dai*, Li Lou, Xingyao Wang, Yilian Huang, Jiao Lei, Feitai Tang, Yangyang Bian, Yong Zeng, Guangxiu Lu, Ge Lin and Shen Zhang*, ","doi":"10.1021/acs.jproteome.5c00440","DOIUrl":"10.1021/acs.jproteome.5c00440","url":null,"abstract":"<p >Calmodulin (CaM) plays a crucial role in sperm function. Studies have reported that proteins containing the IQ motif interact with CaM, subsequently engaging with downstream target proteins known as calmodulin-binding proteins (CaMBPs). However, no relevant reports have been published detailing which CaMBPs exist and the mechanisms by which they are regulated. In this study, we conducted quantitative proteomic and phosphoproteomic analysis for mouse testes from wild-type (WT) and <i>Iqcn</i> knockout (<i>Iqcn</i><sup><i>–/–</i></sup>) mice. The results indicated that <i>Iqcn</i> deficiency substantially rewires the downstream phosphorylation signaling pathway while not causing equivalent changes at protein levels. Among the 577 differentially regulated phosphorylated sites, most of them (494/577) belong to CaMBPs. Gene ontology analysis of these differentially phosphorylated CaMBPs showed enrichment in male gamete generation, actin cytoskeleton organization, and microtubule cytoskeleton organization process, demonstrating that IQCN regulates sperm function by interacting with CaM, which in turn affects the phosphorylation level of CaMBPs. Further kinase-substrate network analysis and the inhibition assay showed that FGFR4 and SYK tyrosine kinases are important for sperm motility and progressive motility. In summary, this study reveals that the interaction between IQCN and CaM regulates the phosphorylation of downstream CaMBPs and is involved in the related processes of spermiogenesis and sperm function.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4699–4707"},"PeriodicalIF":3.6,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144792960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Liangjia Du, Qianqian Hu, Jiaojiao Sha, Zijin Geng, Shanyu Qi, Ting Liu, Huimin Zhu, Dezhu Chen, Minqi Cai, Yiqiang Chen, Siyuan Liu, Hongyan Song and Bing Bai*,
{"title":"Tissue Mild Extraction Enriches Membrane, Cytoplasmic, and Secreted Proteomes for Biomarker Discovery","authors":"Liangjia Du, Qianqian Hu, Jiaojiao Sha, Zijin Geng, Shanyu Qi, Ting Liu, Huimin Zhu, Dezhu Chen, Minqi Cai, Yiqiang Chen, Siyuan Liu, Hongyan Song and Bing Bai*, ","doi":"10.1021/acs.jproteome.5c00494","DOIUrl":"10.1021/acs.jproteome.5c00494","url":null,"abstract":"<p >Clinical blood biomarkers provide important diagnostic information and are generally secreted, cytoplasmic, membrane, and other soluble proteins. However, in the mass spectrometry-based proteomic biomarker discovery phase, the whole tissue is often lysed directly for analysis, and this increases protein complexity in the sample, limiting the detection of soluble proteins as biomarkers. Here, we use a mild extraction method that includes a low salt buffer and 1% Triton X-100 to isolate proteins from mouse tissues followed by proteomic analyses. This mild condition extracts membrane, cytoplasmic, and secreted proteins efficiently while preserving nuclear proteins largely from the mouse brain and 16 other tissues. Further in-depth proteomic analysis of the mild extract from an Alzheimer mouse brain tissue has identified more than 12,000 proteins with enrichments of soluble proteins and potential cerebrospinal fluid markers. This mild extraction has enriched membrane, cytoplasmic, and secreted proteins from tissue samples and should be used as a general method for increased success in the mass spectrometry-based proteomic discovery of circulating biomarkers.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4852–4861"},"PeriodicalIF":3.6,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144792962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Paula Kotli*, David Morgenstern, Shifra Ben-Dor, Liora Kolska Horwitz and Elisabetta Boaretto*,
{"title":"Sex Identification and Species Confirmation in Modern and Archeological Caprine Enamel","authors":"Paula Kotli*, David Morgenstern, Shifra Ben-Dor, Liora Kolska Horwitz and Elisabetta Boaretto*, ","doi":"10.1021/acs.jproteome.5c00012","DOIUrl":"10.1021/acs.jproteome.5c00012","url":null,"abstract":"<p >Proteomics has become a transformative tool for species and sex determination. This study introduces a novel methodology that integrates amelogenin (Amel) and enamelin (Enam) proteins extracted from the tooth enamel of caprines. Since morphologically, osteological remains of sheep and goats often cannot be easily discriminated, we developed our method on both modern domestic sheep (<i>Ovis aries</i>) and goats (<i>Capra hircus</i>) to establish unique proteomic signatures for each species for sex and species identification. Applying a targeted parallel reaction monitoring (PRM) assay, we validated the sex and species of 8 modern domestic sheep and 6 domestic goats. We then applied the same method to 10 ancient samples dating to the early eighth millennium BC Neolithic period. For sex determination, AmelY peptides were exclusively detected in modern male samples, while AmelX peptides were present in both sexes. Sex determination in 10 Neolithic samples demonstrated 40% males. For species determination, Enam species-specific peptides with single amino acid variations (SAAVs) successfully distinguished the modern caprine species. In the 10 archeological samples, only goat-specific Enam peptides were detected, validating previous zooarcheological results for this assemblage using morphology and mtDNA analysis. Robust peptide intensities and strong statistical correlations between modern and ancient data sets confirm the preservation of these unique markers in caprine enamel, expanding the application of proteomics to modern, archeological, and paleontological samples.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4403–4416"},"PeriodicalIF":3.6,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5c00012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144792961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wuping Liu, Yao Huang, Chanyi Li, Ge Song, Mengxiang Xiao, Guiping Shen and Jianghua Feng*,
{"title":"Differential Network Analysis Integrates Pathway Mapping to Characterize Dynamic Metabolic Changes in the Progression of Diabetic Complications","authors":"Wuping Liu, Yao Huang, Chanyi Li, Ge Song, Mengxiang Xiao, Guiping Shen and Jianghua Feng*, ","doi":"10.1021/acs.jproteome.5c00021","DOIUrl":"10.1021/acs.jproteome.5c00021","url":null,"abstract":"<p >Onset and progression of diseases are often characterized by dynamic changes in various metabolites. Monitoring these metabolic fluctuations is a central focus within the field of disease metabolomics. This study introduces an integrative analytical method that combines cross-comparative differential network analysis with network mapping to delineate the dynamic changes of diabetes rats in the fecal metabolome induced by a high-fat diet and streptozotocin. Our results indicate that the fecal metabolite networks are significantly associated with diabetes development. The network analysis identified 13 specific biomarkers linked to the progression of diabetic complications, highlighting that diabetes development is marked by an exacerbation of metabolic dysfunction. Interestingly, the networks analysis also uncovered age-related metabolites including BCAAs (leucine, isoleucine, valine), urocanate, tyrosine, lysine succinate, betaine, and cytosine, which may potentially promote the onset and progression of diabetes. Pathway analysis revealed disruptions in amino acid metabolism, ketone body synthesis and degradation, glycolysis/gluconeogenesis, galactose metabolism, nicotinamide metabolism, and purine metabolism, along with alterations in signaling pathways related to mineral absorption and neurotransmitter synaptic transmission. The cross-comparison network analysis in conjunction with network mapping analysis constitutes an effective method for exploring the dynamic metabolic networks implicated in diseases pathogenesis.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4526–4537"},"PeriodicalIF":3.6,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144783018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Myeloid-Derived Growth Factor-Regulated Oncogenesis in Lung Adenocarcinoma Is Associated with EGFR Status and Cancer Aggressiveness","authors":"Ting-Feng Hsiao, Chih-Liang Wang, Yi-Cheng Wu, Chia-Yu Kuo, Ke-Wei Lin, Wen-Yu Chuang, Chi-Ju Yeh, Chia-Chun Wu, Ko-Jiunn Liu, Gee-Chen Chang, Kun-Yi Chien, Jau-Song Yu and Chia-Jung Yu*, ","doi":"10.1021/acs.jproteome.5c00385","DOIUrl":"10.1021/acs.jproteome.5c00385","url":null,"abstract":"<p >Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have transformed lung adenocarcinoma (LUAD) treatment in EGFR-mutant (MT) patients, but strategies targeting wild-type (WT) EGFR tumors remain necessary. This study analyzed a diverse LUAD patient cohort with EGFR mutation statuses and wild-type profiles for ALK and KRAS to identify stage-specific biomarkers. Using quantitative proteomics and multiomics, we discovered 21 dysregulated proteins in early-stage EGFR-WT LUAD, identifying myeloid-derived growth factor (MYDGF) as a key candidate biomarker. Elevated MYDGF levels in tissue (<i>n</i> = 117) and serum (<i>n</i> = 196) correlated significantly with cancer stage in EGFR-WT patients but not EGFR-MT cases. Notably, a higher tumor-to-normal MYDGF ratio predicted a favorable prognosis in early-stage EGFR-WT LUAD. Functional studies demonstrated that MYDGF exerts distinct roles in cell viability and migration depending on its cellular localization and the invasive potential of cancer cells. Specifically, secreted MYDGF promoted a protumorigenic phenotype, whereas excess intracellular MYDGF appeared to suppress the oncogenic capacity of aggressive cancer cells. MYDGF knockdown and subsequent proteomic analysis provided further insights into these context-dependent functions. These findings highlight EGFR status- and stage-specific proteomic profiles in LUAD, emphasizing the importance of context-dependent biomarker assessment for personalized treatment strategies.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4674–4688"},"PeriodicalIF":3.6,"publicationDate":"2025-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Maowei Ni, Danying Wan, Mengni Li, Junzhou Wu, Juan Ni, Hanmei Lou, Shengjie Zhang* and Zhuomin Yin*,
{"title":"Deficiency of the UBE2C/F264 Axis Underlies Intrinsic Radioresistance in Cervical Carcinoma","authors":"Maowei Ni, Danying Wan, Mengni Li, Junzhou Wu, Juan Ni, Hanmei Lou, Shengjie Zhang* and Zhuomin Yin*, ","doi":"10.1021/acs.jproteome.5c00409","DOIUrl":"10.1021/acs.jproteome.5c00409","url":null,"abstract":"<p >Intrinsic radiotherapy resistance remains a persistent challenge to the treatment of cervical cancer (CC), as the underlying mechanisms remain largely elusive. In this study, proteomic profiling was combined with <i>in vitro</i> and <i>in vivo</i> experiments to clarify the potential mechanisms driving radiotherapy resistance. Initially, Orbitrap Astral-based proteomic profiling of clinical specimens revealed markedly decreased UBE2C expression in radioresistant tumors. Subsequent functional validation with cellular and animal models of UBE2C deficiency revealed that UBE2C is critical for preserving radiosensitivity and its knockdown directly induced the radioresistance of CC. Furthermore, proteomic analysis of UBE2C-knockdown CC cell lines, combined with multilayer intersection analysis of the clinical proteomic data, revealed that the glycolysis-associated protein F264 is potentially regulated by UBE2C. Mechanistically, F264 regulates tumor cell dormancy to evade radiation-induced damage. Finally, immunohistochemical analysis of an independent clinical cohort confirmed aberrant F264 protein expression patterns. Therefore, we hypothesize that altered UBE2C protein levels during cervical carcinogenesis modulate F264 expression, which partially activates tumor dormancy mechanisms, ultimately contributing to the intrinsic radiotherapy resistance observed in a subset of CC patients during initial treatment.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 9","pages":"4665–4673"},"PeriodicalIF":3.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144758722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tobias Zott, Michael Wolf, Günter Plessl-Walder, Heinz Regele, Michael Bergmann, Samuel M. Meier-Menches, Christopher Gerner, Gerd R. Silberhumer and Andrea Bileck*,
{"title":"","authors":"Tobias Zott, Michael Wolf, Günter Plessl-Walder, Heinz Regele, Michael Bergmann, Samuel M. Meier-Menches, Christopher Gerner, Gerd R. Silberhumer and Andrea Bileck*, ","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"24 8","pages":"XXX-XXX XXX-XXX"},"PeriodicalIF":3.6,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/pdf/10.1021/acs.jproteome.5c00114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144748364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}