A systematic investigation into the nature of tryptic HCD spectra

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 our experience and literature knowledge about fragmentation pathways. It aims to automatically annotate the peaks in high mass accuracy fragment spectra while strictly controlling the false discovery rate. Using this Expert System we determined that sequence specific regular ions covering the entire sequence were present for almost all peptides with up to 10 amino acids (median 100%). Peptides up to 20 amino acid length contained sufficient fragmentation to cover 80% of the sequence. Internal fragments are common in HCD spectra but not in high resolution CID spectra (10% vs 1%). The low mass region contains abundant immonium ions (6% of fragment ion intensity), the characteristic a(2), b(2) ion pair (72% of spectra), side chain fragments and reporter ions for peptide modifications such as tyrosine phosphorylation. B- and y-ions account for only 20% of fragment ions by number but 53% by ion intensity. Overall, 84% of the fragment ion intensity was unambiguously explainable. Thus high mass accuracy HCD and CID data are near comprehensively and automatically interpretable.