Use of a Designed Peptide Array To Infer Dissociation Trends for Nontryptic Peptides in Quadrupole Ion Trap and Quadrupole Time-of-Flight Mass Spectrometry

Observed peptide gas-phase fragmentation patterns are
a complex function of many variables. To systematically
probe this phenomenon, an array of 40 peptides was
synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant
and randomize or balance others (peptide amino acid
distribution and position). A high-quality tandem mass
spectrometry (MS/MS) data set was acquired for each
peptide for all observed charge states on multiple MS
instruments, quadrupole-time-of-flight and quadrupole
ion trap. The data were analyzed as a function of total
charge state and number of mobile protons. Previously
known dissociation trends were observed, validating our
approach. In addition, the general influence of basic
amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic
peptides, the amino acids H, K, and R were positionally
distributed. Interestingly, our results suggest that cleavage
at all basic amino acids is suppressed when a mobile
proton is available. Cleavage at H becomes favored only
under conditions where a partially mobile proton is
present, a caveat to the previously reported trend of
enhanced cleavage at H. Finally, all acquired data were
used as a benchmark to determine how well these
sequences would have been identified in a database
search using a common algorithm, Mascot.