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Title: Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells

Abstract

Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of highquality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)- based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels =30 000 and =3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells ormore » in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.« less

Authors:
 [1]; ; ORCiD logo; ; ; ; ; ; ; ; ORCiD logo;  [2];  [2]; ORCiD logo; ORCiD logo; ORCiD logo; ORCiD logo; ; ORCiD logo
  1. Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan 410008, People’s Republic of China
  2. Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland 20892, United States
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1501575
Report Number(s):
PNNL-SA-138130
Journal ID: ISSN 0003-2700
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Analytical Chemistry
Additional Journal Information:
Journal Volume: 91; Journal Issue: 2; Journal ID: ISSN 0003-2700
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English

Citation Formats

Zhang, Pengfei, Gaffrey, Matthew J., Zhu, Ying, Chrisler, William B., Fillmore, Thomas L., Yi, Lian, Nicora, Carrie D., Zhang, Tong, Wu, Huanming, Jacobs, Jon, Tang, Keqi, Kagan, Jacob, Srivastava, Sudhir, Rodland, Karin D., Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Wiley, H. Steven, and Shi, Tujin. Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells. United States: N. p., 2018. Web. doi:10.1021/acs.analchem.8b04258.
Zhang, Pengfei, Gaffrey, Matthew J., Zhu, Ying, Chrisler, William B., Fillmore, Thomas L., Yi, Lian, Nicora, Carrie D., Zhang, Tong, Wu, Huanming, Jacobs, Jon, Tang, Keqi, Kagan, Jacob, Srivastava, Sudhir, Rodland, Karin D., Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Wiley, H. Steven, & Shi, Tujin. Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells. United States. doi:10.1021/acs.analchem.8b04258.
Zhang, Pengfei, Gaffrey, Matthew J., Zhu, Ying, Chrisler, William B., Fillmore, Thomas L., Yi, Lian, Nicora, Carrie D., Zhang, Tong, Wu, Huanming, Jacobs, Jon, Tang, Keqi, Kagan, Jacob, Srivastava, Sudhir, Rodland, Karin D., Qian, Wei-Jun, Smith, Richard D., Liu, Tao, Wiley, H. Steven, and Shi, Tujin. Fri . "Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells". United States. doi:10.1021/acs.analchem.8b04258.
@article{osti_1501575,
title = {Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells},
author = {Zhang, Pengfei and Gaffrey, Matthew J. and Zhu, Ying and Chrisler, William B. and Fillmore, Thomas L. and Yi, Lian and Nicora, Carrie D. and Zhang, Tong and Wu, Huanming and Jacobs, Jon and Tang, Keqi and Kagan, Jacob and Srivastava, Sudhir and Rodland, Karin D. and Qian, Wei-Jun and Smith, Richard D. and Liu, Tao and Wiley, H. Steven and Shi, Tujin},
abstractNote = {Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of highquality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)- based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels =30 000 and =3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.},
doi = {10.1021/acs.analchem.8b04258},
journal = {Analytical Chemistry},
issn = {0003-2700},
number = 2,
volume = 91,
place = {United States},
year = {2018},
month = {11}
}