Predicting Peptide Retention Times for Proteomics

Oleg V. Krokhin1, Vic Spicer2

1 Department of Internal Medicine, University of Manitoba, Winnipeg, Canada, 2 Department of Physics and Astronomy, University of Manitoba, Winnipeg, Canada
Publication Name:  Current Protocols in Bioinformatics
Unit Number:  Unit 13.14
DOI:  10.1002/0471250953.bi1314s31
Online Posting Date:  September, 2010
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The vast majority of modern bottom‐up proteomic protocols include chromatographic reversed‐phase (RP) fractionation of peptides prior to mass‐spectrometric analysis. Retention time information can be easily extracted from LC‐MS data and it can be used to improve protein identification/characterization procedures. The key to the success of this procedure is the correct retention time prediction based on compositional and structural properties of the separated species. Our Sequence Specific Retention Calculator (SSRCalc) is a Web‐based peptide retention prediction that covers the separation selectivity of the most popular RP‐HPLC conditions applied in proteomics. Procedures for the application of SSRCalc to proteomic analyses are described in this unit. Curr. Protoc. Bioinform. 31:13.14.1‐13.14.15. © 2010 by John Wiley & Sons, Inc.

Keywords: peptide retention prediction; reversed‐phase HPLC; protein identification

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Predicting Peptide Retention Times for Filtering MS/MS Identifications
  • Basic Protocol 2: Predicting Peptide Retention Times for De‐Novo Development of Scheduled MRM/SRM Procedures
  • Guidelines for Understanding Results
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Predicting Peptide Retention Times for Filtering MS/MS Identifications

  • Mass‐spectrometer capable of performing MRM/SRM analysis
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Literature Cited

   Baczek, T., Wiczling, P., Marszall, M., Heyden, Y.V., and Kaliszan, R. 2005. Prediction of peptide retention at different HPLC conditions from multiple linear regression models. J. Proteome Res. 4:555‐563.
   Browne, C.A., Bennett, H.P., and Solomon, S. 1982. The isolation of peptides by high‐performance liquid chromatography using predicted elution positions. Anal Biochem. 124:201‐208.
   Dwivedi, R.C., Spicer, V., Harder, M., Antonovici, M., Ens, W., Standing, K.G., Wilkins, J.A., and Krokhin, O.V. 2008. Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high‐throughput bottom‐up proteomics. Anal Chem. 80:7036‐7042.
   Gorshkov, A.V., Tarasova, I.A., Evreinov, V.V., Savitski, M.M., Nielsen, M.L., Zubarev, R.A., and Gorshkov, M.V. 2006. Liquid chromatography at critical conditions: Comprehensive approach to sequence‐dependent retention time prediction. Anal Chem. 78:7770‐7777.
   Guo, D., Mant, C.T., Taneja, A.K., Parker, R., and Hodges, R.S. 1986. Prediction of peptide retention times in reversed‐phase high‐performance liquid chromatography. I. Determination of retention coefficients of amino acid residues of model synthetic peptides. J. Chromatogr. 359:499‐517.
   Krokhin, O.V. 2006. Sequence‐specific retention calculator. Algorithm for peptide retention prediction in ion‐pair RP‐HPLC: Application to 300‐ and 100‐A pore size C18 sorbents. Anal Chem. 78:7785‐7795.
   Krokhin, O.V. and Spicer, V. 2009. Peptide retention standards and hydrophobicity indexes in reversed‐phase high‐performance liquid chromatography of peptides. Anal Chem. 81:9522‐9530.
   Krokhin, O.V., Ens, W., and Standing, K.G. 2003. Characterizing degradation products of peptides containing N‐terminal Cys residues by (off‐line high‐performance liquid chromatography)/matrix‐assisted laser desorption/ionization quadrupole time‐of‐flight measurements. Rapid Commun. Mass Spectrom. 17:2528‐2534.
   Krokhin, O.V., Craig, R., Spicer, V., Ens, W., Standing, K.G., Beavis, R.C., and Wilkins, J.A. 2004. An improved model for prediction of retention times of tryptic peptides in ion pair reversed‐phase HPLC: Its application to protein peptide mapping by off‐line HPLC‐MALDI MS. Mol. Cell Proteomics 3:908‐919.
   Lange, V., Picotti, P., Domon, B., and Aebersold, R. 2008. Selected reaction monitoring for quantitative proteomics: A tutorial. Mol. Syst. Biol. 4:222.
   Mead, J.A., Bianco, L., Ottone, V., Barton, C., Kay, R.G., Lilley, K.S., Bond, N.J., and Bessant, C. 2009. MRMaid, the web‐based tool for designing multiple reaction monitoring (MRM) transitions. Mol. Cell Proteomics 8:696‐705.
   Meek, J.L. 1980. Prediction of peptide retention times in high‐pressure liquid chromatography on the basis of amino acid composition. Proc. Natl. Acad. Sci. U.S.A. 3:1632‐1636.
   Petritis, K., Kangas, L.J., Yan, B., Monroe, M.E., Strittmatter, E.F., Qian, W.J., Adkins, J.N., Moore, R.J., Xu, Y., Lipton, M.S., Camp, D.G. 2nd, and Smith, R.D. 2006. Improved peptide elution time prediction for reversed‐phase liquid chromatography‐MS by incorporating peptide sequence information. Anal Chem. 78:5026‐5039.
   Shinoda, K., Sugimoto, M., Yachie, N., Sugiyama, N., Masuda, T., Robert, M., Soga, T., and Tomita, M. 2006. Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome using artificial neural networks. J. Proteome Res. 5:3312‐3317.
   Spicer, V., Yamchuk, A., Cortens, J., Sousa, S., Ens, W., Standing, K. G., Wilkins, J.A., and Krokhin, O.V. 2007. Sequence‐specific retention calculator. A family of peptide retention time prediction algorithms in reversed‐phase HPLC: Applicability to various chromatographic conditions and columns. Anal Chem. 79:8762‐8768.
   Strittmatter, E.F., Kangas, L.J., Petritis, K., Mottaz, H.M., Anderson, G.A., Shen, Y., Jacobs, J.M., Camp, D.G. 2nd, and Smith, R.D. 2004. Application of peptide LC retention time information in a discriminant function for peptide identification by tandem mass spectrometry. J. Proteome Res. 3:760‐769.
Key References
   Krokhin, 2006. See above.
  The most complete description of the SSRCalc model.
   Krokhin et al., 2004. See above.
  First version of the SSRCalc model.
   Krokhin and Spicer, 2009. See above.
  Introduction of standard P1‐P6 peptide mixture and new hydrophobicity scales.
Internet Resources
  SSRCalc online version.
  TheorChromo online version.
  NET Prediction Utility download site.
Supplemental Data
  Mascot Generic Format file: 101208‐mix25‐075‐1.mgf
  Sample file for the LC‐MS/MS analysis of the tryptic digest mixture of four human proteins spiked with a 6‐peptide standard mixture ().
  Excel file: 101208‐mix25‐075‐1.xls
  Contains the list of all identified peptides (including non‐tryptic peptides and post‐translationally modified ones), as well as a non‐redundant list of tryptic unmodified species.
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