Protein Digestion by Endoproteinase AspN for Improved Localization of Protein Modifications by Peptide End-specific Marker Ions

摘要

Reversible protein modification is the central principle for regulation of many cellular functions and therefore of high analytical interest. Tandem mass spectrometry is a powerful tool for protein identification and modification analysis. However, it is a common phenomenon that a substantial portion of peptide MS/MS spectra is left unannotated by search engine-assisted data interpretation. Covalent modifications not considered in data interpretation probably are a major cause for this failure. The limitations of an automated search for covalent modifications currently include that the type of modification has to be preselected and that only a small number of modifications can be searched simultaneously. Here, a strategy for assignment of covalently modified peptides is presented, based on assignment of end-specific fragment ions. Endoproteinase AspN cleaves proteins at the N-terminal side of Asp and Glu residues. Thus, the N-termini of AspN-peptides are formed by one of only 38 possible dipeptide sequences with predefined composition and sequence direction (19 starting with D, 19 starting E, assuming that Leu and Ile cannot be distinguished and considering oxidized Met in addition). Thus, the complete variability of AspN-generated b_(2) ions is covered by about 40 'golden' ions. The mass values of these b_(2) ions can be reliably recognized by the mass accuracy provided by a Q-TOF instrument [1]. The significance of b_(2) ions is normally supported by a_(2) ions and additional fragments, and their discrimination from internal dipeptide b ions can be accomplished, e.g. by their complementary y_(max-2) ions.