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Molecular & Cellular Proteomics 3:908-919, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

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^*

O.V. Krokhin^,,¶, R. Craig, V. Spicer, W. Ens^,, K. G. Standing^,, R. C. Beavis and J. A. Wilkins

Department of Physics and Astronomy, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada; and Manitoba Centre for Proteomics, University of Manitoba, Winnipeg, MB, R3E 3P4, Canada

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R² value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier.

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