Journal of Proteome Research最新文献_第5页

Improved Method to Determine Protein Turnover Rates with Heavy Water Labeling by Mass Isotopomer Ratio Selection.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-18 DOI: 10.1021/acs.jproteome.4c01012

Jordan Currie, Dominic C M Ng, Boomathi Pandi, Alexander Black, Vyshnavi Manda, Cheyanne Durham, Jay Pavelka, Maggie P Y Lam, Edward Lau

{"title":"Improved Method to Determine Protein Turnover Rates with Heavy Water Labeling by Mass Isotopomer Ratio Selection.","authors":"Jordan Currie, Dominic C M Ng, Boomathi Pandi, Alexander Black, Vyshnavi Manda, Cheyanne Durham, Jay Pavelka, Maggie P Y Lam, Edward Lau","doi":"10.1021/acs.jproteome.4c01012","DOIUrl":"10.1021/acs.jproteome.4c01012","url":null,"abstract":"The synthesis and degradation rates of proteins form an essential component of gene expression control. Heavy water labeling has been used in conjunction with mass spectrometry to measure protein turnover rates, but the optimal analytical approaches to derive turnover rates from the mass isotopomer patterns of deuterium-labeled peptides continue to be a subject of research. Here, we describe a method that comprises (1) a nearest lookup of numerically approximated peptide isotope envelopes, coupled to (2) the selection of optimal mass isotopomer pairs based on peptide sequence rules, to calculate the molar fraction of new peptide synthesis in heavy water labeling mass spectrometry experiments. We validated our approach using an experimental calibration standard comprising mixtures of fully unlabeled and fully labeled proteomes. We then reanalyzed 17 proteome-wide turnover experiments from four mouse organs across multiple data sets and showed that the combined nearest-lookup and rule-based mass isotopomer ratio selection method increases the coverage of well-fitted peptides in protein turnover experiments by up to 58 ± 13%. The workflow is implemented in the Riana software tool for protein turnover analysis and may avail ongoing efforts to study the synthesis and degradation kinetics of proteins in animals on a proteome-wide scale.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1992-2005"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143655515","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}

引用次数: 0

HYTANE-Identified Latrophilin-3 Cleavage by Meprin β Leads to Loss of the Interaction Domains.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-26 DOI: 10.1021/acs.jproteome.4c00912

Fred Armbrust, Kira Bickenbach, Tomas Koudelka, Corentin Joos, Maximilian Keller, Andreas Tholey, Claus U Pietrzik, Christoph Becker-Pauly

{"title":"HYTANE-Identified Latrophilin-3 Cleavage by Meprin β Leads to Loss of the Interaction Domains.","authors":"Fred Armbrust, Kira Bickenbach, Tomas Koudelka, Corentin Joos, Maximilian Keller, Andreas Tholey, Claus U Pietrzik, Christoph Becker-Pauly","doi":"10.1021/acs.jproteome.4c00912","DOIUrl":"10.1021/acs.jproteome.4c00912","url":null,"abstract":"The metalloprotease meprin β is upregulated in neurons and astrocytes of Alzheimer's disease patients' brains. While the role of meprin β as the β-secretase of amyloid precursor protein (APP) has been characterized, its broader substrate profile within the brain remains largely unexplored. Hence, to identify additional substrates, we conducted N-terminomics of brain lysates from mice overexpressing meprin β in astrocytes employing the Hydrophobic Tagging-Assisted N-terminal Enrichment (HYTANE) strategy. We observed 3906 (82.2%) N-terminal peptides and identified seven new substrates that match meprin β in terms of localization and cleavage specificity. Of note, the meprin β overexpressing mice show mild cognitive impairments caused by amyloidogenic APP processing alongside hyperactivity and altered exploratory behavior seemingly independent of APP cleavage. Hence, latrophilin-3 was of particular interest, as latrophilin-3 defects are associated with hyperactivity in mice and human. In brain lysates from mice overexpressing meprin β in astrocytes as well as in cellulo, we validated the cleavage of latrophilin-3, resulting in the release of two N-terminal domains. These domains promote interactions with neuronal proteins such as fibronectin leucine-rich repeat transmembrane proteins, promoting adequate synapse formation. Thus, meprin β might affect synaptic integrity by cleaving interaction domains of latrophilin-3, potentially exacerbating the observed hyperactivity phenotype.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1832-1844"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707765","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}

引用次数: 0

Antioxidant Impact of Soft Knotwood Extracts on Human Keratinocytes Shown by NMR Metabolomic Analysis.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-26 DOI: 10.1021/acs.jproteome.4c00836

Océane Quin, Marylène Bertrand, Pauline Gerardin, Philippe Gerardin, Christine Gerardin-Charbonnier, Céline Landon, Chantal Pichon

{"title":"Antioxidant Impact of Soft Knotwood Extracts on Human Keratinocytes Shown by NMR Metabolomic Analysis.","authors":"Océane Quin, Marylène Bertrand, Pauline Gerardin, Philippe Gerardin, Christine Gerardin-Charbonnier, Céline Landon, Chantal Pichon","doi":"10.1021/acs.jproteome.4c00836","DOIUrl":"10.1021/acs.jproteome.4c00836","url":null,"abstract":"The Pinophyta family has long been used to protect the skin from oxidation, thanks to the action of molecules such as stilbenes, flavonoids, and lignans, which are particularly concentrated in knotwood. These molecules are of interest from a cosmetic perspective. The present study focuses on four species from larch (Larix decidua Mill.), silver fir (Abies alba Mill.), Norway spruce (Picea abies (L.) H.Karst), and Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) knotwood, recovered from byproducts of the wood industry. The molecules are extracted from knotwood and used in vitro on human keratinocytes (HaCaT). Studies quantifying reactive oxygen species (ROS) have demonstrated its ability to eliminate hydroxyl radicals and superoxides. Metabolomic analyses using proton nuclear magnetic resonance (1H NMR) and multivariate statistics (PLS-DA) demonstrated that keratinocytes modulate metabolite expression after treatment with knot extracts. Indeed, our findings indicate an increase in metabolites such as glutathione, glycine, glutamate, sarcosine, taurine, and proline, which are known to reduce intracellular oxidative stress and validate the effect on ROS levels. They also indicate that knotwood extracts may affect membrane balance, collagen formation, and oxidative stress levels. This study highlights the value of metabolomic analysis in the cosmetic industry for a detailed understanding of the mechanisms implemented in a whole cell.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1745-1756"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143707756","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}

引用次数: 0

EasyPubPlot: A Shiny Web Application for Rapid Omics Data Exploration and Visualization.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-07 DOI: 10.1021/acs.jproteome.4c01068

Nguyen Tran Nam Tien, Nguyen Quang Thu, Dong Hyun Kim, Seongoh Park, Nguyen Phuoc Long

{"title":"EasyPubPlot: A Shiny Web Application for Rapid Omics Data Exploration and Visualization.","authors":"Nguyen Tran Nam Tien, Nguyen Quang Thu, Dong Hyun Kim, Seongoh Park, Nguyen Phuoc Long","doi":"10.1021/acs.jproteome.4c01068","DOIUrl":"10.1021/acs.jproteome.4c01068","url":null,"abstract":"Computational toolkits for data exploration and visualization from widely used omics platforms often lack flexibility and customization. While many tools generate standardized output, advanced programming skills are necessary to create high-quality visualizations. Therefore, user-friendly tools that simplify this crucial, yet time-consuming, step are essential. We developed EasyPubPlot (Easy Publishable Plotting), a straightforward, easy-to-use, no-coding, user experience-oriented, open-source, and shiny web application along with its associated R package to streamline data exploration and visualization for functional omics-empowered research. EasyPubPlot generates publishable scores plots, volcano plots, heatmaps, box plots, dot plots, and bubble plots with minimal necessary steps. The tool was designed to guide new users to accurate and efficient navigation. Step-by-step tutorials for each type of plot are also provided. Herein, we demonstrated EasyPubPlot's competent functionality and versatility by showcasing metabolomics, proteomics, and transcriptomics data. Collectively, EasyPubPlot reduces the gap between data analysis and stunning visualization, thereby diminishing friction and focusing on science. The app can be downloaded and installed locally (https://github.com/Pharmaco-OmicsLab/EasyPubPlot) or used through a web application (https://pharmaco-omicslab.shinyapps.io/EasyPubPlot).","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"2188-2195"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143575607","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}

引用次数: 0

Single Cell Untargeted Lipidomics Using Liquid Chromatography Ion Mobility-Mass Spectrometry.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-02-14 DOI: 10.1021/acs.jproteome.4c00658

Jinyong Kim, Dong-Gi Mun, Husheng Ding, Erica Marie Forsberg, Sven W Meyer, Aiko Barsch, Akhilesh Pandey, Seul Kee Byeon

{"title":"Single Cell Untargeted Lipidomics Using Liquid Chromatography Ion Mobility-Mass Spectrometry.","authors":"Jinyong Kim, Dong-Gi Mun, Husheng Ding, Erica Marie Forsberg, Sven W Meyer, Aiko Barsch, Akhilesh Pandey, Seul Kee Byeon","doi":"10.1021/acs.jproteome.4c00658","DOIUrl":"10.1021/acs.jproteome.4c00658","url":null,"abstract":"Advancements in technology over the years have propelled omics analysis to the level of single cell resolution. Following the breakthroughs in single cell transcriptomics and genomics, single cell proteomics has recently rapidly progressed, aided by highly sensitive mass spectrometry instrumentation. However, there is currently a paucity of studies and methodologies for single cell lipidomics, aside from imaging-based approaches. Profiling lipids at the single cell level holds promise for providing novel insights into the complex heterogeneity of cells in various human disorders. Further, by integrating single cell lipidomics with other single cell omics including proteomics, it becomes possible to achieve single cell multiomics, enabling the discovery of novel molecular signatures. We developed untargeted single cell lipidomics using nanoflow liquid chromatography-ion mobility spectrometry-mass spectrometry. To enhance lipid coverage at the single cell level, the method was conducted in both positive and negative ion modes. We identified an average of 161 lipids spanning phospholipids, sphingolipids, cholesteryl esters, and glycerides in positive ion mode from single cells of human cholangiocarcinoma cells based on a rule-based lipid annotation. Additionally, an average of 20 species of phospholipids was identified in the negative ion mode. These preliminary data demonstrate a new methodology to profile lipids at a single or low input of cells.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1579-1585"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143412374","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}

引用次数: 0

Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-02 DOI: 10.1021/acs.jproteome.4c01040

Lia R Serrano, Adrian Pelin, Tabiwang N Arrey, Nicolaie E Damoc, Alicia L Richards, Yuan Zhou, Noah M Lancaster, Trenton M Peters-Clarke, Anna Pashkova, Gwendolyn M Jang, Manon Eckhardt, Scott T Quarmby, Martin Zeller, Daniel Hermanson, Hamish Stewart, Christian Hock, Alexander Makarov, Vlad Zabrouskov, Nevan J Krogan, Joshua J Coon, Danielle L Swaney

{"title":"Affinity Purification Mass Spectrometry on the Orbitrap-Astral Mass Spectrometer Enables High-Throughput Protein-Protein Interaction Mapping.","authors":"Lia R Serrano, Adrian Pelin, Tabiwang N Arrey, Nicolaie E Damoc, Alicia L Richards, Yuan Zhou, Noah M Lancaster, Trenton M Peters-Clarke, Anna Pashkova, Gwendolyn M Jang, Manon Eckhardt, Scott T Quarmby, Martin Zeller, Daniel Hermanson, Hamish Stewart, Christian Hock, Alexander Makarov, Vlad Zabrouskov, Nevan J Krogan, Joshua J Coon, Danielle L Swaney","doi":"10.1021/acs.jproteome.4c01040","DOIUrl":"10.1021/acs.jproteome.4c01040","url":null,"abstract":"Classical proteomics experiments offer high-throughput protein quantification but lack direct evidence of the spatial organization of the proteome, including protein-protein interaction (PPIs) networks. While affinity purification mass spectrometry (AP-MS) is the method of choice for generating these networks, technological impediments have stymied the throughput of AP-MS sample collection and therefore constrained the rate and scale of experiments that can be performed. Here, we build on advances in mass spectrometry hardware that have rendered high-flow liquid chromatography separations a viable solution for faster throughput quantitative proteomics. We describe our methodology using the Orbitrap-Astral mass spectrometer with 7 min, high-flow separations to analyze 216 AP-MS samples in ∼29 h. We show that the ion-focusing advancements, rapid mass analysis, and sensitive ion detection facilitate narrow-bin data-independent acquisition on a chromatographically practical timescale. Further, we highlight several aspects of state-of-the-art confidence-scoring software that warrant reinvestigation given the analytical characteristics of the Orbitrap-Astral mass spectrometer through comparisons with an enrichment-based thresholding technique. With our data, we generated an interaction map between 998 human proteins and 59 viral proteins. These results hold promise in expediting the throughput of AP-MS experiments, enabling more high-powered PPI studies.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"2006-2016"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536155","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}

引用次数: 0

Proteomics and Metabolomics Analyses Reveal a Dynamic Landscape of Coal Workers' Pneumoconiosis: An Insight into Disease Progression.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-04 DOI: 10.1021/acs.jproteome.4c00715

Yangyang Wei, Zhenzhen Jia, Jing Ma, Wei Zhang, Hui Li, Juan Wu, Xiaojing Wang, Xiao Yu, Yiwei Shi, Xiaomei Kong, Min Pang

{"title":"Proteomics and Metabolomics Analyses Reveal a Dynamic Landscape of Coal Workers' Pneumoconiosis: An Insight into Disease Progression.","authors":"Yangyang Wei, Zhenzhen Jia, Jing Ma, Wei Zhang, Hui Li, Juan Wu, Xiaojing Wang, Xiao Yu, Yiwei Shi, Xiaomei Kong, Min Pang","doi":"10.1021/acs.jproteome.4c00715","DOIUrl":"10.1021/acs.jproteome.4c00715","url":null,"abstract":"Coal worker's pneumoconiosis (CWP) is characterized by chronic inflammation and pulmonary fibrosis. The key factor contributing to the incurability of CWP is the unclear pathogenesis. This study explored the characteristic changes in proteomics and metabolomics of early and advanced CWP patients through proteomics and metabolomics techniques. Proteomics identified proteins that change with the progression of CWP, with significant enrichment in the TGF-β signaling pathway and autoimmune disease pathways. Metabolomics revealed the metabolic characteristics of CWP at different stages. These metabolites mainly include changes in amino acid metabolism, unsaturated fatty acid synthesis, and related metabolites. Integrated analysis found that ABC transporters are a shared pathway among the three groups, and ABCD2 is involved in the ABC transporter pathway. In the subsequent independent sample verification analysis, consistent with proteomics experiments, compared to the CM group, FMOD expression level was upregulated in the NIC group. TFR expression level was consistently downregulated in both the IC and NIC groups. Additionally, ABCD2 increased in the IC group but decreased in the NIC group. In summary, this study revealed the metabolic characteristics of CWP at different stages. These findings may provide valuable insights for the early prediction, diagnosis, and treatment of CWP.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1715-1731"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143555275","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}

引用次数: 0

SpecPeptidOMS Directly and Rapidly Aligns Mass Spectra on Whole Proteomes and Identifies Peptides That Are Not Necessarily Tryptic: Implications for Peptidomics.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2025-03-27 DOI: 10.1021/acs.jproteome.4c00870

Émile Benoist, Géraldine Jean, Hélène Rogniaux, Guillaume Fertin, Dominique Tessier

{"title":"SpecPeptidOMS Directly and Rapidly Aligns Mass Spectra on Whole Proteomes and Identifies Peptides That Are Not Necessarily Tryptic: Implications for Peptidomics.","authors":"Émile Benoist, Géraldine Jean, Hélène Rogniaux, Guillaume Fertin, Dominique Tessier","doi":"10.1021/acs.jproteome.4c00870","DOIUrl":"10.1021/acs.jproteome.4c00870","url":null,"abstract":"SpecPeptidOMS directly aligns peptide fragmentation spectra to whole and undigested protein sequences. The algorithm was specifically and initially designed for peptidomics, where the aim is to identify peptides that do not result from the hydrolysis of a known protein and therefore, whose termini cannot be predicted. Thus, SpecPeptidOMS can perform alignments starting and ending anywhere in the protein sequence. The underlying computational method of SpecPeptidOMS, which is based on a dynamic programming approach, was drastically optimized. As a result, SpecPeptidOMS can process around 12,000 spectra per hour on an ordinary laptop, with alignment performed against the entire human proteome. The performance of SpecPeptidOMS was first evaluated on a publicly available data set of (nontryptic) synthetic mass spectra. Accuracy was estimated by considering the results obtained by MaxQuant on the same data set as the \"ground truth\". A second series of tests on a larger, well-known proteomics data set (HEK293) highlighted SpecPeptidOMS' additional ability to search for open modifications, a feature of interest in peptidomics but also more broadly in conventional proteomics. SpecPeptidOMS is open-source, cross-platform (written in Java), and freely available.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"2159-2172"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717667","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}

引用次数: 0

Comparison of Protein Solubilization and Normalization Methods for Proteomics Analysis of Extracellular Vesicles from Urine.

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 DOI: 10.1021/acs.jproteome.4c01085

Jun Wang, Yang Li, Yisheng Wang, Guoli Wang, Chenyang Zhao, Ying Zhang, Haojie Lu

{"title":"Comparison of Protein Solubilization and Normalization Methods for Proteomics Analysis of Extracellular Vesicles from Urine.","authors":"Jun Wang, Yang Li, Yisheng Wang, Guoli Wang, Chenyang Zhao, Ying Zhang, Haojie Lu","doi":"10.1021/acs.jproteome.4c01085","DOIUrl":"https://doi.org/10.1021/acs.jproteome.4c01085","url":null,"abstract":"Extracellular vesicles (EVs) play a vital role in numerous biological processes. Proteomic research of EVs is crucial for understanding their functions and potential therapeutic implications. Despite many sample preparation protocols for mass spectrometry-based proteomics of EVs being described, the variability in protein extraction across different protocols has not been extensively investigated. Moreover, given the inherent heterogeneity of EVs, it is vital to conduct a thorough evaluation of normalization methods. Here, we present a comprehensive comparison of three widely used lysis agents─sodium dodecyl sulfate (SDS), urea, and sodium deoxycholate (SDC)─for protein extraction from EVs. We also assess the impact of different normalization strategies on protein quantification, which is crucial for ensuring reliable results. Our results show that method-dependent differences in protein recovery were observed, particularly for membrane-associated proteins. We also find that common normalization strategies, such as urine creatinine and EV markers, did not significantly stabilize protein quantification, indicating that these methods are not universally applicable as normalization standards. Our work thereby provides a reference for the selection of MS sample preparation and normalization strategies for a given EV proteomics project.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784369","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}

引用次数: 0

Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry. 独立数据采集将毛细管电泳质谱的单细胞蛋白质组学分析窗口缩短至十五分钟

IF 3.8 2区生物学

Journal of Proteome Research Pub Date : 2025-04-04 Epub Date: 2024-09-26 DOI: 10.1021/acs.jproteome.4c00491

Bowen Shen, Leena R Pade, Peter Nemes

{"title":"Data-Independent Acquisition Shortens the Analytical Window of Single-Cell Proteomics to Fifteen Minutes in Capillary Electrophoresis Mass Spectrometry.","authors":"Bowen Shen, Leena R Pade, Peter Nemes","doi":"10.1021/acs.jproteome.4c00491","DOIUrl":"10.1021/acs.jproteome.4c00491","url":null,"abstract":"Separation in single-cell mass spectrometry (MS) improves molecular coverage and quantification; however, it also elongates measurements, thus limiting analytical throughput to study large populations of cells. Here, we advance the speed of bottom-up proteomics by capillary electrophoresis (CE) high-resolution mass spectrometry (MS) for single-cell proteomics. We adjust the applied electrophoresis potential to readily control the duration of electrophoresis. On the HeLa proteome standard, shorter separation times curbed proteome detection using data-dependent acquisition (DDA) but not data-independent acquisition (DIA) on an Orbitrap analyzer. This DIA method identified 1161 proteins vs 401 proteins by the reference DDA within a 15 min effective separation from single HeLa-cell-equivalent (∼200 pg) proteome digests. Label-free quantification found these exclusively DIA-identified proteins in the lower domain of the concentration range, revealing sensitivity improvement. The approach also significantly advanced the reproducibility of quantification, where ∼76% of the DIA-quantified proteins had <20% coefficient of variation vs ∼43% by DDA. As a proof of principle, the method allowed us to quantify 1242 proteins in subcellular niches in a single, neural-tissue fated cell in the live Xenopus laevis (frog) embryo, including many canonical components of organelles. DIA integration enhanced throughput by ∼2-4 fold and sensitivity by a factor of ∼3 in single-cell (subcellular) CE-MS proteomics.","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":"1549-1559"},"PeriodicalIF":3.8,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11936843/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142337364","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}

引用次数: 0