Conserved peptide fragmentation as a benchmarking tool for mass spectrometers and a discriminating feature for...

Quantifying the similarity of spectra is an important task in various areas of spectroscopy, for example, to identify a compound by comparing sample spectra to those of reference standards. In mass spectrometry based discovery proteomics, spectral comparisons are used to infer the amino acid sequence of peptides. In targeted proteomics by selected reaction monitoring (SRM) or SWATH MS, predetermined sets of fragment ion signals integrated over chromatographic time are used to identify target peptides in complex samples. In both cases, confidence in peptide identification is directly related to the quality of spectral matches. In this study, we used sets of simulated spectra of well-controlled dissimilarity to benchmark different spectral comparison measures and to develop a robust scoring scheme that quantifies the similarity of fragment ion spectra. We applied the normalized spectral contrast angle score to quantify the similarity of spectra to objectively assess fragment ion variability of tandem mass spectrometric datasets, to evaluate portability of peptide fragment ion spectra for targeted mass spectrometry across different types of mass spectrometers and to discriminate target assays from decoys in targeted proteomics. Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure. The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface.