Methods for peptide identification by spectral comparison

Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies.

This paper investigates computational issues related to this spectral comparison approach. Different methods have been empirically evaluated over several large sets of spectra. First, we illustrate that the peak intensities follow a Poisson distribution. This implies that applying a square root transform will optimally stabilize the peak intensity variance. Our results show that the square root did indeed outperform other transforms, resulting in improved accuracy of spectral matching. Second, different measures of spectral similarity were compared, and the results illustrated that the correlation coefficient was most robust. Finally, we examine how to assemble multiple spectra associated with the same peptide to generate a synthetic reference spectrum. Ensemble averaging is shown to provide the best combination of accuracy and efficiency.

Our results demonstrate that when combined, these methods can boost the sensitivity and specificity of spectral comparison. Therefore they are capable of enhancing and complementing existing tools for consistent and accurate peptide identification.