The Peptide MS/MS-Fragmentome: A Set of Predictable Fragment Ions with Highly Redundant Sequence Information

Extended Abstract Low-energy collision-induced dissociation (CID) of peptides in a collision cell has been introduced as a fast method for peptide sequencing [1]. The method has been adapted to the analysis of gel-isolated proteins by the introduction of the nanoESI technology [2]. In the meantime proteins can be identified by search-engine supported interpretation of raw data of peptide MS/MS spectra in correlation to a protein sequence database [e.g. 3]. A triplet of information, consisting of the protease specificity (mostly trypsin), the molecular weight of the precursor peptide, and a series of MS/MS sequence ions usually forms the basic information for search engine-supported protein identification in sequence databases by ESIMS/MS data. In spite of the success of this approach, in practical work the majority of automatically acquired ESI-MS/MS spectra of tryptic peptides is often left unidentified. Unknown covalent modifications, incorrect assignment of charge states of precursor and fragment ions, the absence of a sufficiently long series of sequence ions, or the selection of non-optimal instrumental conditions are considered to be the main reasons for this situation. Here, we focus on the fact that besides the highly significant y ion series, MS/MS spectra of multiply charged peptides contain a wealth of sequence information in a variety of fragment ion types. Since peptide fragmentation is severely sequence-dependent, knowledge of all sequence-specific features of a peptide MS/MS spectrum is highly useful. An individual peptide may present a certain part of its sequence only in a single type of fragmentation, for instance only by neutral loss fragmentations or by internal fragments. In this case, the sequence information would be overlooked by considering exclusively y ions. On the other hand, the recognition of redundant sequence information contained in several types of fragmentations is also highly useful, since this phenomenon increases the reliability of the sequence assignment. Moreover, redundancy of sequence information may enable an otherwise impossible clear decision in case more than one precursor ion is (automatically) selected in the first stage of MS/MS. Using the phosphopeptide SA-pT-PEALAFVR as an example, a description of the redundant structural information is provided that can be extracted from the peptide MS/MS-fragmentome, which is the universe of MS/MS fragments of multiply charged peptide ions.