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Modern mass spectrometry-based proteomics can produce millions of peptide fragmentation spectra, which are automatically identified in databases using sequence-specific b- or y-ions. Proteomics projects have mainly been performed with low resolution collision-induced dissociation (CID) in ion traps and beam-type fragmentation on triple quadrupole and QTOF instruments. Recently, the latter has also become available with Orbitrap instrumentation as higher energy collisional dissociation (HCD), routinely providing full mass range fragmentation with high mass accuracy. To systematically study the nature of HCD spectra, we made use of a large scale data set of tryptic peptides identified with an FDR of 0.0001, from which we extract a subset of more than 16 000 that have little or no contribution from cofragmented precursors. We employed a newly developed computer-assisted “Expert System”, which distills …