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Direct digestion of proteins in living cells into peptides for proteomic analysis

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Abstract

To analyze the proteome of an extremely low number of cells or even a single cell, we established a new method of digesting whole cells into mass-spectrometry-identifiable peptides in a single step within 2 h. Our sampling method greatly simplified the processes of cell lysis, protein extraction, protein purification, and overnight digestion, without compromising efficiency. We used our method to digest hundred-scale cells. As far as we know, there is no report of proteome analysis starting directly with as few as 100 cells. We identified an average of 109 proteins from 100 cells, and with three replicates, the number of proteins rose to 204. Good reproducibility was achieved, showing stability and reliability of the method. Gene Ontology analysis revealed that proteins in different cellular compartments were well represented.

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Acknowledgments

This work was supported by the National High-Tech R&D Program of China (project 2012AA020202), the National Basic Research Program of China (projects 2012CB910604, 2013CB911201), and the National Natural Science Foundation of China (project 21175026).

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Authors and Affiliations

  1. Department of Chemistry, Institute of Biomedical Sciences, Fudan University, Shanghai, 200433, China

    Qi Chen, Guoquan Yan, Mingxia Gao & Xiangmin Zhang

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  1. Qi Chen
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  2. Guoquan Yan
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  3. Mingxia Gao
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  4. Xiangmin Zhang
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Correspondence to Xiangmin Zhang.

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Published in the topical collection celebrating ABCs 13th Anniversary.

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Chen, Q., Yan, G., Gao, M. et al. Direct digestion of proteins in living cells into peptides for proteomic analysis. Anal Bioanal Chem 407, 1027–1032 (2015). https://doi.org/10.1007/s00216-014-8173-1

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  • DOI: https://doi.org/10.1007/s00216-014-8173-1

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