The commonly used methods for assigning peptide sequences to MS/MS spectra are critically discussed and compared, from basic strategies to advanced multi-stage approaches.

This manuscript provides a comprehensive review of the peptide and protein identification process using tandem mass spectrometry (MS/MS) data generated in shotgun proteomic experiments. We also demonstrate that iterative search strategies applied to such detected unassigned high quality spectra significantly increase the number of spectra that can be assigned from datasets and that biologically interesting new insights can be gained from existing data. To increase the amount of information that can be extracted from proteomic MS/MS datasets we developed a robust method that detects high quality spectra within the fraction of spectra unassigned by conventional sequence database searching and computes a quality score for each spectrum. splice forms, somatic rearrangement, and processed proteins). single nucleotide polymorphisms) or omissions in the database searched, post-translational or chemical modifications of the peptide analyzed, or the observation of sequences that are not anticipated from the genomic sequence (e.g. These include deficiencies of the scoring schemes implemented in the database search tools, sequence variations (e.g. Spectra are not assigned to peptides for diverse reasons. Of these, a relatively small fraction is confidently assigned to peptide sequences, whereas the majority of the spectra are not further analyzed. In mass spectrometry-based proteomics, frequently hundreds of thousands of MS/MS spectra are collected in a single experiment.

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