Abstract
Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.
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Acknowledgments
This work was supported by the China State Key Basic Research Program Grant (2013CB911202, 2012CB910101, and 2012CB910604), the Creative Research Group Project of NSFC (21321064), the National Natural Science Foundation of China (21275142, 21235006, 81161120540, and 81361128015), National Key Special Program on Infection diseases (2012ZX10002009-011), and Analytical Method Innovation Program of MOST (2012IM030900).
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Pan, Y., Cheng, K., Mao, J. et al. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion. Anal Bioanal Chem 406, 6247–6256 (2014). https://doi.org/10.1007/s00216-014-8071-6
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DOI: https://doi.org/10.1007/s00216-014-8071-6