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\begin{document}

\title{\emph{N}-terminal chemical derivatization of peptides with
4-formyl-benzenesulfonic acid under charge reduction conditions and
electrospray positive and negative tandem mass spectrometry \emph{de
novo} sequencing}



\author[1]{Luka Ozdanovac}%
\author[1]{Lucija Dončević}%
\author[1]{Amela Hozić}%
\author[1]{Renata Biba}%
\author[2]{Ema Svetličić}%
\author[3]{Andrea Janeš}%
\author[1]{Mario Cindrić}%
\affil[1]{Ruđer Bošković Institute}%
\affil[2]{Technical University of Denmark}%
\affil[3]{Clinical hospital "Sveti Duh"}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\begin{abstract}
\textbf{RATIONALE:} Selective derivatization of peptide
\emph{N}-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables
chemically activated fragmentation in ESI+/- (positive and negative ion
mode) under charge reduction conditions. Overlapped positive and
negative tandem mass spectra pinpoint b-ions making the assignment of
b-ion series fragments easy and accurate. \textbf{METHODS:} We developed
a FBSA-peptide microwave assisted derivatization procedure. Derivatized
and non-derivatized bovine serum albumin tryptic peptides and insulin
non-tryptic peptide were compared after MS/MS analysis in positive and
negative ion mode. High-quality dataset 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.
\textbf{RESULTS:} The FBSA derivatization procedure produced a
significantly improved MS/MS dataset (populated by high-intensity
signals of b- and y-ions) in comparison to commonly used
\emph{N}-terminal sulfonation reagents. Undesired side reactions do not
occur and the procedure reduces the derivatization time. It was found
that b-ions comprise 15\% and 13\% of all fragment ions generated in
positive and negative ion mode, respectively. High visibility of b-ion
series in negative ion mode can be attributed to the \emph{N}-terminal
sulfonation which had no effect on the production of b-ion series in
positive ion mode. \textbf{CONCLUSIONS:} The FBSA derivatization and
\emph{de novo} sequencing approach outlined here provides a reliable
method for accurate peptide sequence assignment. Increased production of
the b-ions in positive and negative ion mode greatly improves peak
assignment and thus enables accurate sequence reconstruction.
Implementation of the named methodology would improve the quality of
\emph{de novo} sequencing data and reduce the number of misinterpreted
spectra.%
\end{abstract}%



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