Use of Peptide Retention Time Prediction for Protein Identification by off-line Reversed-Phase HPLC−MALDI MS/MS

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Abstract
A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-Å pore size columns. Correlations of up to ∼0.98 R2 value were obtained for a test library of ∼2000 peptides and ∼0.95−0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC−MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.