Skip to main content

Advertisement

SpringerLink
Log in

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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.

Sequence logos of four cleavage site types with different kinetics (very fast, fast, slow, and very slow sites)

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Hunt DF, Yates JR, Shabanowitz J, Winston S, Hauer CR (1986) Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci U S A 83(17):6233–6237

    Article  CAS  Google Scholar 

  2. Wu C, Tran JC, Zamdborg L, Durbin KR, Li M, Ahlf DR, Early BP, Thomas PM, Sweedler JV, Kelleher NL (2012) A protease for ‘middle-down’ proteomics. Nat Methods 9(8):822–824

    Article  CAS  Google Scholar 

  3. Olsen JV, Ong S-E, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3(6):608–614

    Article  CAS  Google Scholar 

  4. Siepen JA, Keevil E-J, Knight D, Hubbard SJ (2007) Prediction of missed cleavage sites in tryptic peptides aids protein identification in proteomics. J Proteome Res 6(1):399–408

    Article  CAS  Google Scholar 

  5. Lawless C, Hubbard SJ (2012) Prediction of missed proteolytic cleavages for the selection of surrogate peptides for quantitative proteomics. Omics 16(9):449–456

    Article  CAS  Google Scholar 

  6. Gershon PD (2013) Cleaved and missed sites for trypsin, Lys-C and Lys-N can be predicted with high confidence on the basis of sequence context. J Proteome Res 13(2):702–709

    Article  Google Scholar 

  7. Wang S-S, Carpenter FH (1968) Kinetic studies at high pH of the trypsin-catalyzed hydrolysis of N α-benzoyl derivatives of l-arginamide, l-lysinamide, and S-2-aminoethyl-l-cysteinamide and related compounds. J Biol Chem 243(13):3702–3710

    CAS  Google Scholar 

  8. Simpson B, Haard N (1984) Purification and characterization of trypsin from the Greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics. Can J Biochem Cell Biol 62(9):894–900

    Article  CAS  Google Scholar 

  9. Caprioli RM, Smith L (1986) Determination of Km and Vmax for tryptic peptide hydrolysis using fast atom bombardment mass spectrometry. Anal Chem 58(6):1080–1083

    Article  CAS  Google Scholar 

  10. Fraser D, Powell RE (1950) The kinetics of trypsin digestion. J Biol Chem 187:803–820

    CAS  Google Scholar 

  11. Halsey JF, Harrington WF (1973) Substructure of paramyosin. Correlation of helix stability, trypsin digestion kinetics, and amino acid composition. Biochemistry 12(4):693–701

    Article  CAS  Google Scholar 

  12. Walmsley SJ, Rudnick PA, Liang Y, Dong Q, Stein SE, Nesvizhskii AI (2013) Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics. J Proteome Res 12(12):5666–5680

    Article  CAS  Google Scholar 

  13. Ye M, Pan Y, Cheng K, Zou H (2014) Protein digestion priority is independent of protein abundances. Nat Methods 11(3):220–222

    Article  CAS  Google Scholar 

  14. Boersema PJ, Raijmakers R, Lemeer S, Mohammed S, Heck AJR (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4(4):484–494

    Article  CAS  Google Scholar 

  15. Bian Y, Ye M, Song C, Cheng K, Wang C, Wei X, Zhu J, Chen R, Wang F, Zou H (2012) Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach. J Proteome Res 11(5):2828–2837

    Article  CAS  Google Scholar 

  16. Song C, Wang F, Ye M, Cheng K, Chen R, Zhu J, Tan Y, Wang H, Figeys D, Zou H (2011) Improvement of the quantification accuracy and throughput for phosphoproteome analysis by a pseudo triplex stable isotope dimethyl labeling approach. Anal Chem 83(20):7755–7762

    Article  CAS  Google Scholar 

  17. Wang F, Chen R, Zhu J, Sun D, Song C, Wu Y, Ye M, Wang L, Zou H (2010) A fully automated system with online sample loading, isotope dimethyl labeling and multidimensional separation for high-throughput quantitative proteome analysis. Anal Chem 82(7):3007–3015

    Article  CAS  Google Scholar 

  18. Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372

    Article  CAS  Google Scholar 

  19. Crooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14(6):1188–1190

    Article  CAS  Google Scholar 

  20. Rodriguez J, Gupta N, Smith RD, Pevzner PA (2007) Does trypsin cut before proline? J Proteome Res 7(1):300–305

    Article  Google Scholar 

  21. Yang S, Nie A, Zhang L, Yan G, Yao J, Xie L, Lu H, Yang P (2012) A novel quantitative proteomics workflow by isobaric terminal labeling. J Proteome 75(18):5797–5806

    Article  CAS  Google Scholar 

  22. Li Z, Adams RM, Chourey K, Hurst GB, Hettich RL, Pan C (2012) Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap velos. J Proteome Res 11(3):1582–1590

    Article  CAS  Google Scholar 

  23. Schechter I, Berger A (1967) On the size of the active site in proteases. I Papain. Biochem Biophys Res Commun 27(2):157–162

    Article  CAS  Google Scholar 

  24. Thiede B, Lamer S, Mattow J, Siejak F, Dimmler C, Rudel T, Jungblut PR (2000) Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion. Rapid Commun Mass Spectrom 14(6):496–502

    Article  CAS  Google Scholar 

  25. Switzar L, Giera M, Niessen WM (2013) Protein digestion: an overview of the available techniques and recent developments. J Proteome Res 12(3):1067–1077

    Article  CAS  Google Scholar 

  26. Apweiler R, Biswas M, Fleischmann W, Kanapin A, Karavidopoulou Y, Kersey P, Kriventseva EV, Mittard V, Mulder N, Phan I (2001) Proteome analysis database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes. Nucleic Acids Res 29(1):44–48

    Article  CAS  Google Scholar 

  27. Keil B (1992) Specificity of proteolysis. Springer, Berlin

    Book  Google Scholar 

  28. Liu H, Sadygov RG, Yates JR (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76(14):4193–4201

    Article  CAS  Google Scholar 

  29. Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR, Resing KA, Ahn NG (2005) Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4(10):1487–1502

    Article  CAS  Google Scholar 

  30. Ning K, Fermin D, Nesvizhskii AI (2012) Comparative analysis of different label-free mass spectrometry based protein abundance estimates and their correlation with RNA-Seq gene expression data. J Proteome Res 11(4):2261–2271

    Article  CAS  Google Scholar 

  31. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132

    Article  CAS  Google Scholar 

  32. Atsushi I (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88(6):1895–1898

    Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Yanbo Pan, Kai Cheng, Jiawei Mao, Fangjie Liu, Jing Liu, Mingliang Ye & Hanfa Zou

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yanbo Pan, Kai Cheng, Jiawei Mao, Fangjie Liu & Jing Liu

Authors
  1. Yanbo Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kai Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiawei Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fangjie Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jing Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mingliang Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hanfa Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Mingliang Ye or Hanfa Zou.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 1,980 kb)

ESM 2

(XLS 1.97 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-014-8071-6

Keywords

Search

Navigation