Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn |
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| Authors: | Linhui Zhai Cheng Chang Ning Li Duc M Duong Hao Chen Zixin Deng Jian Yang Xuechuan Hong Yunping Zhu Ping Xu |
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| Institution: | 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;3. Tianjin Institute of Medical Equipment, , Tianjin, P. R. China |
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| 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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| Keywords: | Hydrophobicity Microcolumn MS Quantitative proteomics SILAC Technology |
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