Confident Identification of Citrullination and Carbamylation Assisted by Peptide Retention Time Prediction

The
chromatographic behavior of peptides carrying citrulline and
homocitrulline residues in proteomic two-dimensional (2D) liquid chromatography–mass
spectrometry (LC–MS) experiments has been investigated. The
primary goal of this study was to determine the chromatographic conditions
that allow differentiating between arginine citrullination and deamidation
of asparagine based on retention data, improving the confidence of
MS-based identifications. Carbamylation was used as a reference point
due to a high degree of similarity between modification products and
anticipated changes in chromatographic behavior. We applied 2D LC–MS/MS
(a high-pH–low-pH reversed phase (RP), hydrophilic interaction
liquid chromatography (HILIC)–low-pH RP, and strong cation
exchange (SCX)–low-pH RP) to acquire retention data for modified–nonmodified
peptide pairs in the four separation modes. Modifications of a standard
protein mixture were induced enzymatically (PAD-2) or chemically (urea)
for citrullination and carbamylation, respectively. Deamidation occurs
spontaneously. Similar retention shifts were observed for all three
modifications in a high-pH RP (decrease) and a low-pH RP (increase),
thus limiting the applicability of this 2D LC combination. HILIC on
bare silica and strong cation exchange separations have been probed
to amplify the effect of charge loss upon citrullination, with SCX
demonstrating the most differentiating power: the elimination of basic
residues upon citrullination/carbamylation results in an ∼58
mM KCl retention decrease, while retention of deamidated products
decreases slightly.