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

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

doi:10.1021/acs.analchem.8b04258

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

Journal Article · Mon Dec 17 00:00:00 EST 2018 · Analytical Chemistry

DOI:https://doi.org/10.1021/acs.analchem.8b04258· OSTI ID:1501575

Zhang, Pengfei ^[1]; Gaffrey, Matthew J. ^[2]; ^[3]; Chrisler, William B. ^[2]; Fillmore, Thomas L. ^[3]; Yi, Lian ^[2]; Nicora, Carrie D. ^[2]; Zhang, Tong ^[2]; Wu, Huanming ^[2]; Jacobs, Jon ^[2]; ^[2]; Kagan, Jacob ^[4]; Srivastava, Sudhir ^[4]; ^[2]; ^[2]; ^[2]; ^[2]; Wiley, H. Steven ^[3]; ^[2]

Central South Univ., Changsha, HN (People’s Republic of China); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
National Cancer Inst. (NCI), Bethesda, MD (United States)

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. In this paper 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.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Organization:: China Scholarship Council (CSC); National Cancer Institute (NCI); National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1501575

Report Number(s):: PNNL-SA--138130

Journal Information:: Analytical Chemistry, Journal Name: Analytical Chemistry Journal Issue: 2 Vol. 91; ISSN 0003-2700

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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