RT‐PSM, a real‐time program for peptide‐spectrum matching with statistical significance

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Abstract
The analysis of complex biological peptide mixtures by tandem mass spectrometry (MS/MS) produces a huge body of collision‐induced dissociation (CID) MS/MS spectra. Several methods have been developed for identifying peptide‐spectrum matches (PSMs) by assigning MS/MS spectra to peptides in a database. However, most of these methods either do not give the statistical significance of PSMs (e.g., SEQUEST) or employ time‐consuming computational methods to estimate the statistical significance (e.g., PeptideProphet). In this paper, we describe a new algorithm, RT‐PSM, which can be used to identify PSMs and estimate their accuracy statistically in real time. RT‐PSM first computes PSM scores between an MS/MS spectrum and a set of candidate peptides whose masses are within a preset tolerance of the MS/MS precursor ion mass. Then the computed PSM scores of all candidate peptides are employed to fit the expectation value distribution of the scores into a second‐degree polynomial function in PSM score. The statistical significance of the best PSM is estimated by extrapolating the fitting polynomial function to the best PSM score. RT‐PSM was tested on two pairs of MS/MS spectrum datasets and protein databases to investigate its performance. The MS/MS spectra were acquired using an ion trap mass spectrometer equipped with a nano‐electrospray ionization source. The results show that RT‐PSM has good sensitivity and specificity. Using a 55 577‐entry protein database and running on a standard Pentium‐4, 2.8‐GHz CPU personal computer, RT‐PSM can process peptide spectra on a sequential, one‐by‐one basis in 0.047 s on average, compared to more than 7 s per spectrum on average for Sequest and X!Tandem, in their current batch‐mode processing implementations. RT‐PSM is clearly shown to be fast enough for real‐time PSM assignment of MS/MS spectra generated every 3 s or so by a 3D ion trap or by a QqTOF instrument. Copyright © 2006 John Wiley & Sons, Ltd.