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Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post‐translational modification analysis
Author(s) -
Chen Rui,
Stupak Jacek,
Williamson Sam,
Twine Susan M.,
Li Jianjun
Publication year - 2019
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.8459
Subject(s) - chemistry , chromatography , tandem mass spectrometry , mass spectrometry , phosphopeptide , peptide , biochemistry
Rationale Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed‐phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site‐specific glycosylation. Methods Digests of protein standards (horse myoglobin, bovine fetuin and β‐casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2‐Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 μm i.d. × 100 mm) packed with 3 μm particles. MS/MS data were collected in data‐dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. Results PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from β‐casein by efficient separation of the phosphopeptide and its non‐phosphorylated counterpart, which cannot be achieved by reversed‐phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2‐3‐ and α2‐6‐linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. Conclusions PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post‐translational modifications (PTMs) by polar groups.