Dataset Information

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

ABSTRACT: Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 ?g of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.

SUBMITTER: Mertins P

PROVIDER: S-EPMC6211289 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

Mertins Philipp P Tang Lauren C LC Krug Karsten K Clark David J DJ Gritsenko Marina A MA Chen Lijun L Clauser Karl R KR Clauss Therese R TR Shah Punit P Gillette Michael A MA Petyuk Vladislav A VA Thomas Stefani N SN Mani D R DR Mundt Filip F Moore Ronald J RJ Hu Yingwei Y Zhao Rui R Schnaubelt Michael M Keshishian Hasmik H Monroe Matthew E ME Zhang Zhen Z Udeshi Namrata D ND Mani Deepak D Davies Sherri R SR Townsend R Reid RR Chan Daniel W DW Smith Richard D RD Zhang Hui H Liu Tao T Carr Steven A SA

Nature protocols 20180701 7

Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two ...[more]

PMID: 29988108

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Project description:<p>Mertins P, Tang LC, Krug K, Clark DJ, Gritsenko MA, Chen L, Clauser KR, Clauss TR, Shah P, Gillette MA, et al., <a href="https://www.nature.com/articles/s41596-018-0006-9" target="_blank">Nat Protoc. 2018 Jul 9. doi: 10.1038/s41596-018-0006-9</a></p><p>Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)10) for multiplexed analysis and relative quantification, and provides 3x higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 ug of peptide derived from <50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was <7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was <24% across laboratories, with an average of >37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in ~4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.</p><ul><li>Dataset imported into MassIVE from <a href="https://cptac-data-portal.georgetown.edu/cptac/s/S036">https://cptac-data-portal.georgetown.edu/cptac/s/S036</a> on 08/30/19</li></ul>

Dataset Information

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

Publications

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

Similar Datasets

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