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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

  • Kosuke Hata
    Kosuke Hata
    Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
    More by Kosuke Hata
  • Yoshihiro Izumi*
    Yoshihiro Izumi
    Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
    *E-mail: [email protected]. Tel: +81-92-642-6171.
  • Takeshi Hara
    Takeshi Hara
    Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
    More by Takeshi Hara
  • Masaki Matsumoto*
    Masaki Matsumoto
    Division of Proteomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
    Department of Omics and Systems Biology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757, Asahimachi-dori, Niigata, 951-8510, Japan
    *E-mail: [email protected]. Tel: +81-25-227-2077.
  • , and 
  • Takeshi Bamba
    Takeshi Bamba
    Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
Cite this: Anal. Chem. 2020, 92, 4, 2997–3005
Publication Date (Web):January 21, 2020
https://doi.org/10.1021/acs.analchem.9b03993
Copyright © 2020 American Chemical Society

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    Abstract

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    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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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.9b03993.

    • Details of cell culture and harvesting; details of conventional sample preparation; and details of preparation of the immobilized trypsin column and nano-LC column (PDF)

    • Sample preparation methods; peptides and proteins identified from the blank and from HeLa cells; and relationship between the peak area and number of cells for peptides (XLSX)

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    Cited By

    This article is cited by 11 publications.

    1. Lingxiao Weng, Guoquan Yan, Wei Liu, Qunfei Tai, Mingxia Gao, Xiangmin Zhang. Picoliter Single-Cell Reactor for Proteome Profiling by In Situ Cell Lysis, Protein Immobilization, Digestion, and Droplet Transfer. Journal of Proteome Research 2024, Article ASAP.
    2. Shuang Yang, Yueting Xiong, Yang Du, Ya-Jun Wang, Lei Zhang, Fenglin Shen, Yan-Jun Liu, Xiaohui Liu, Pengyuan Yang. Ultrasensitive Trace Sample Proteomics Unraveled the Protein Remodeling during Mesenchymal–Amoeboid Transition. Analytical Chemistry 2022, 94 (2) , 768-776. https://doi.org/10.1021/acs.analchem.1c03212
    3. Yuanyuan Li, Hang Li, Yuping Xie, Shuo Chen, Ritian Qin, Hangyan Dong, Yongliang Yu, Jianhua Wang, Xiaohong Qian, Weijie Qin. An Integrated Strategy for Mass Spectrometry-Based Multiomics Analysis of Single Cells. Analytical Chemistry 2021, 93 (42) , 14059-14067. https://doi.org/10.1021/acs.analchem.0c05209
    4. Kendall Martin, Tong Zhang, Tai-Tu Lin, Amber N. Habowski, Rui Zhao, Chia-Feng Tsai, William B. Chrisler, Ryan L. Sontag, Daniel J. Orton, Yong-Jie Lu, Karin D. Rodland, Bin Yang, Tao Liu, Richard D. Smith, Wei-Jun Qian, Marian L. Waterman, H. Steven Wiley, Tujin Shi. Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50–1000 Mammalian Cells. Journal of Proteome Research 2021, 20 (9) , 4452-4461. https://doi.org/10.1021/acs.jproteome.1c00403
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    6. Sooyeon KIM, Latiefa KAMARULZAMAN, Yuichi TANIGUCHI. Recent methodological advances towards single-cell proteomics. Proceedings of the Japan Academy, Series B 2023, 99 (8) , 306-327. https://doi.org/10.2183/pjab.99.021
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    9. Cynthia Nagy, Ruben Szabo, Attila Gaspar. Microfluidic Immobilized Enzymatic Reactors for Proteomic Analyses—Recent Developments and Trends (2017–2021). Micromachines 2022, 13 (2) , 311. https://doi.org/10.3390/mi13020311
    10. Hanne Røberg-Larsen, Elsa Lundanes, Tuula A. Nyman, Frode S. Berven, Steven Ray Wilson. Liquid chromatography, a key tool for the advancement of single-cell omics analysis. Analytica Chimica Acta 2021, 1178 , 338551. https://doi.org/10.1016/j.aca.2021.338551
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