Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Authors

  • Linhui Zhai,

    1. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
    2. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, P. R. China
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    • These authors contributed equally to this work.

  • Cheng Chang,

    1. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
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    • These authors contributed equally to this work.

  • Ning Li,

    Corresponding author
    • State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
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  • Duc M. Duong,

    1. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
    Current affiliation:
    1. Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, GA, USA
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  • Hao Chen,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, P. R. China
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  • Zixin Deng,

    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, P. R. China
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  • Jian Yang,

    1. Tianjin Institute of Medical Equipment, Tianjin, P. R. China
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  • Xuechuan Hong,

    Corresponding author
    • Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, P. R. China
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  • Yunping Zhu,

    Corresponding author
    • State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
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  • Ping Xu

    Corresponding author
    1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, P. R. China
    • State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Engineering Research Center for Protein Drugs, National Center for Protein Sciences, Beijing Institute of Radiation Medicine, Beijing, P. R. China
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  • Colour Online: See the article online to view Figs. 1–4 in colour.

Correspondence: Professor Ping Xu, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, P. R. China

E-mail: xupingghy@gmail.com

Fax: +8610-80705155

Additional corresponding authors:

Dr. Ning Li, E-mail: pkuningonly@gmail.com

Professor Xuechuan Hong, E-mail: xhy78@whu.edu.cn

Professor Yunping Zhu, E-mail: zhuyunping@gmail.com

Abstract

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic-binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic-binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.

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