N ‐Terminal chemical derivatization of peptides with 4‐formyl‐benzenesulfonic acid and electrospray positive and negative tandem mass spectrometry with high abundance of b‐ion series

Rationale: Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge reduction conditions. Overlapped positive and negative tandem mass spectra show b-ions making the assignment of b-ion series fragments easy and accurate.// Methods: We developed an FBSA–peptide microwave-assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high-quality data set of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences.// Results: The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high-intensity signals of b- and y-ions) compared to commonly used N-terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b-ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive- and negative ion modes, respectively. High visibility of b- ion series in negative ion mode can be attributed to N-terminal sulfonation that had no negative effect on the production of b- and y-ion series in positive ion mode.// Conclusions: The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b-ions in positive- and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.