TY - JOUR
T1 - In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells
AU - Hata, Kosuke
AU - Izumi, Yoshihiro
AU - Hara, Takeshi
AU - Matsumoto, Masaki
AU - Bamba, Takeshi
N1 - Funding Information:
This study was supported by the JST-CREST Program (JPMJCR15G4) of the Japan Science and Technology Agency (JST) (Y.I., M.M., and T.B.), a Grant-in-Aid for Scientific Research on Innovative Areas (17H06304) from the Japan Society for the Promotion of Science (JSPS) (Y.I. and T.B.), a Grant-in-Aid for Scientific Research on Innovative Areas (17H06011, 19H04969, and 19H05243) from JSPS (M.M.), and a Grant-in-Aid for Scientific Research (C) (19K05167) from JSPS (Y.I.).
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/2/18
Y1 - 2020/2/18
N2 - Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.
AB - Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.
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U2 - 10.1021/acs.analchem.9b03993
DO - 10.1021/acs.analchem.9b03993
M3 - Article
AN - SCOPUS:85080914272
VL - 92
SP - 2997
EP - 3005
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 4
ER -