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Combination of online enzyme digestion with stable isotope labeling for high-throughput quantitative proteome analysis

Authors

  • Fangjun Wang,

    1. CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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  • Xiaoluan Wei,

    1. CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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  • Hu Zhou,

    1. Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada
    2. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
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  • Jing Liu,

    1. CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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  • Daniel Figeys,

    1. Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Canada
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  • Hanfa Zou

    Corresponding author
    • CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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  • Colour Online: See the article online to view Figs. 1, 3 and 4 in colour.

Correspondence: Professor Hanfa Zou, CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

E-mail: hanfazou@dicp.ac.cn

Fax: +86-411-84379620

Abstract

Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post-digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two-fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 105 Hela cells).

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