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Updated: Jan 25, 2026

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry
Published on: January 5, 2021
Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post-translational modification
Rui Chen1, Jacek Stupak1, Sam Williamson1
1Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada.
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.
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