Individual b2 ion fragmentation profiles combined with AspN digestion improve N-terminal peptide sequencing.

The N terminus of peptides generated by AspN is restricted to about 40 dipeptide motifs starting with D or E. These motifs are visible upon collision-induced dissociation (CID) as b(2) ions, which are often the most abundant low-mass fragment ions. It was observed that b(2) ions are accompanied by a set of sequence-specific neutral losses of CO, H(2)O, NH(3), and some other small units. To test the utility of these profiles as additional parameters for reliable assignment of the b(2) ion motif besides its m/z value, the CID spectra of 221 different AspN-generated peptides covering all N-terminal D-X and E-X motifs were recorded. Qualitatively, the b(2) ion fragmentation profiles of individual motifs were found to exhibit little dependency on the rest of the peptide sequence. Thus, it is concluded that the set of b(2) ion fragmentation profiles recorded in this study can be used as reference set. Knowledge of these profiles provides an increased specificity for b(2) ion annotation of AspN-generated peptides compared to the use of only a solitary b(2) ion m/z value. Recognition of the b(2) ion motif provides a two-amino-acid sequence including its direction; it provides the location of this motif at the N terminus, and it sets a starting point for further extension of the b ion series.