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Title: 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 intraand 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 µg 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 interlaboratory 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 formore » 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.« less

Authors:
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Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1503571
Report Number(s):
PNNL-SA-137930
Journal ID: ISSN 1754-2189
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Nature Protocols
Additional Journal Information:
Journal Volume: 13; Journal Issue: 7; Journal ID: ISSN 1754-2189
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English

Citation Formats

Mertins, Philipp, Tang, Lauren C., Krug, Karsten, Clark, David J., Gritsenko, Marina A., Chen, Lijun, Clauser, Karl R., Clauss, Therese R., Shah, Punit, Gillette, Michael A., Petyuk, Vladislav A., Thomas, Stefani N., Mani, D. R., Mundt, Filip, Moore, Ronald J., Hu, Yingwei, Zhao, Rui, Schnaubelt, Michael, Keshishian, Hasmik, Monroe, Matthew E., Zhang, Zhen, Udeshi, Namrata D., Mani, Deepak, Davies, Sherri R., Townsend, R. Reid, Chan, Daniel W., Smith, Richard D., Zhang, Hui, Liu, Tao, and Carr, Steven A. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry. United States: N. p., 2018. Web. doi:10.1038/s41596-018-0006-9.
Mertins, Philipp, Tang, Lauren C., Krug, Karsten, Clark, David J., Gritsenko, Marina A., Chen, Lijun, Clauser, Karl R., Clauss, Therese R., Shah, Punit, Gillette, Michael A., Petyuk, Vladislav A., Thomas, Stefani N., Mani, D. R., Mundt, Filip, Moore, Ronald J., Hu, Yingwei, Zhao, Rui, Schnaubelt, Michael, Keshishian, Hasmik, Monroe, Matthew E., Zhang, Zhen, Udeshi, Namrata D., Mani, Deepak, Davies, Sherri R., Townsend, R. Reid, Chan, Daniel W., Smith, Richard D., Zhang, Hui, Liu, Tao, & Carr, Steven A. Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry. United States. doi:10.1038/s41596-018-0006-9.
Mertins, Philipp, Tang, Lauren C., Krug, Karsten, Clark, David J., Gritsenko, Marina A., Chen, Lijun, Clauser, Karl R., Clauss, Therese R., Shah, Punit, Gillette, Michael A., Petyuk, Vladislav A., Thomas, Stefani N., Mani, D. R., Mundt, Filip, Moore, Ronald J., Hu, Yingwei, Zhao, Rui, Schnaubelt, Michael, Keshishian, Hasmik, Monroe, Matthew E., Zhang, Zhen, Udeshi, Namrata D., Mani, Deepak, Davies, Sherri R., Townsend, R. Reid, Chan, Daniel W., Smith, Richard D., Zhang, Hui, Liu, Tao, and Carr, Steven A. Sun . "Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry". United States. doi:10.1038/s41596-018-0006-9.
@article{osti_1503571,
title = {Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography–mass spectrometry},
author = {Mertins, Philipp and Tang, Lauren C. and Krug, Karsten and Clark, David J. and Gritsenko, Marina A. and Chen, Lijun and Clauser, Karl R. and Clauss, Therese R. and Shah, Punit and Gillette, Michael A. and Petyuk, Vladislav A. and Thomas, Stefani N. and Mani, D. R. and Mundt, Filip and Moore, Ronald J. and Hu, Yingwei and Zhao, Rui and Schnaubelt, Michael and Keshishian, Hasmik and Monroe, Matthew E. and Zhang, Zhen and Udeshi, Namrata D. and Mani, Deepak and Davies, Sherri R. and Townsend, R. Reid and Chan, Daniel W. and Smith, Richard D. and Zhang, Hui and Liu, Tao and Carr, Steven A.},
abstractNote = {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 intraand 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 µg 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 interlaboratory 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.},
doi = {10.1038/s41596-018-0006-9},
journal = {Nature Protocols},
issn = {1754-2189},
number = 7,
volume = 13,
place = {United States},
year = {2018},
month = {7}
}

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