Differential ¹⁴N/¹⁵N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing

抄録

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.