Neutral loss of isocyanic acid in peptide CID spectra: a novel diagnostic marker for mass spectrometric identification of protein citrullination

Abstract

Protein citrullination is emerging as an important signaling mechanism that modulates a variety of biological processes. This protein modification constitutes only a 1 Da mass shift, and can be readily confused with other common protein modifications that yield an identical mass shift. In an attempt to develop a robust methodology for detection of protein citrullination sites, we analyzed synthetic citrulline-containing peptides by electrospray ionization tandem mass spectrometry. Collision-induced dissociation (CID) spectra revealed abundant neutral loss of 43 Da from citrullinated peptide precursor ions, which was reconciled by elimination of the HNCO moiety (isocyanic acid) from the citrulline ureido group. The elimination occurs readily in multiple charge states of precursor ions and also in b and y ions. HNCO loss in CID spectra provides a novel diagnostic marker for citrullination, and its utility was demonstrated by the discovery of Arg197 as the specific site of citrullination on nucleophosmin upon peptidylarginine deiminase 4 treatment.

Figure 1

Identification of citrullination site in PAD-4 treated human nucleophosmin (NPM). Protein bands were in-gel digested with trypsin and resulting peptides were analyzed by LC-MS. Data-dependant MS/MS scans were performed on the two most intense ions in each MS scan. The CID spectra were searched for neutral loss of 43 and 21.5 Da. A single peptide ion, corresponding to SI{Cit}DTPAK containing Arg197 was found in PAD4-treated band. The observed m/z was 888.4 for the singly-charged peptide ion and 444.7 for the doubly-charged species. The CE used for the peptide fragmentation was calculated using Analyst software (42 and 28 for singly- and doubly-charged ions, respectively.) Panel (a), Total ion chromatograms (TIC) of PAD4-treated and untreated human NPM. The CID spectra were extracted to generate neutral loss spectra of 43 and 21.5 Da. Shown here is the 43 Da neutral loss chromatogram that reveals a singly charged peptide SI{Cit}DTPAK eluted around 12 min in the PAD4-treated sample. The neutral loss chromatogram of 21.5 Da similarly identified the doubly charged precursor of the same citrullinated peptide (not shown). Panel (b) and (c), CID spectra of the citrullinated peptide SI{Cit}DTPAK and the native peptide SIRDTPAK. HNCO loss was abundant in CID spectra of both the singly and doubly charged citrullinated peptide ions, whereas only ammonia loss was found in the unmodified peptide spectra.

Scheme 1

Proposed fragmentation mechanism for isocyanic acid elimination from citrulline peptides