While
de novo peptide sequencing is essential in many situations, it remains a difficult task. This is because peptide fragmentation results in complicated and often incomplete product ion spectra. In a previous study, we demonstrated that N-terminal charge derivatization with 4-amidinobenzoic acid (Aba) resulted in improved peptide fragmentation under low-energy CID conditions. However, even with this derivatization, some ambiguity exists, due to difficulties in discriminating between N- and C-terminal fragments. In this study, to specifically identify b-ions from complex product ion spectra, the differential
14N/
15N-labeling of peptides was performed using Aba derivatization.
15N-Labeled Aba was synthesized in the form of a succinimide ester. Peptides were derivatized individually with
14N-Aba or
15N-Aba and analyzed by ESI-MS/MS using a linear ion trap-Orbitrap hybrid FTMS system. The N-terminal fragments (
i.e., b-ions) were then identified based on
m/
z differences arising from isotope labeling. By comparing the spectra between
14N- and
15N-Aba derivatized peptides, b-ions could be successfully identified based on the
m/
z shifts, which provided reliable sequencing results for all of the peptides examined in this study. The method developed in this study allows the easy and reliable
de novo sequencing of peptides, which is useful in peptidomics and proteomics studies.
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