title: Identification and quantification of glycoproteins using ion-pairing normal-phase liquid chromatography and mass spectrometry.
abstract: Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures. Here we describe that the use of acids as ion-pairing reagents in normal-phase chromatography (IP-NPLC) considerably increases the hydrophobicity differences between non-glycopeptides and glycopeptides, thereby resulting in the reproducible isolation of N-linked high mannose type and sialylated glycopeptides from the tryptic digest of a ribonuclease B and fetuin mixture. The elution order of non-glycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. O-linked glycopeptides can be efficiently isolated from fetuin tryptic digests using IP-NPLC when N-glycans are first removed with PNGase. IP-NPLC recovers close to 100% of bacterial N-linked glycopeptides modified with non-sialylated heptasaccharides from tryptic digests of periplasmic protein extracts from Campylobacter jejuni 11168 and its pglD mutant. Label-free nano-flow reversed-phase LC-MS is used for quantification of differentially expressed glycopeptides from the C. jejuni wild-type and pglD mutant followed by identification of these glycoproteins using multiple stage tandem MS. This method further confirms the acetyltransferase activity of PglD and demonstrates for the first time that heptasaccharides containing monoacetylated bacillosamine are transferred to proteins in both the wild-type and mutant strains. We believe that IP-NPLC will be a useful tool for quantitative glycoproteomics.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada. wen.ding@nrc-cnrc.gc.ca
authors.forename: Wen
authors.initials: W
authors.lastname: Ding
authors.forename: Harald
authors.initials: H
authors.lastname: Nothaft
authors.forename: Christine M
authors.initials: CM
authors.lastname: Szymanski
authors.forename: John
authors.initials: J
authors.lastname: Kelly
mesh_terms: D000143:Acids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016123:Campylobacter jejuni / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019158:N-Acetylneuraminic Acid; D012015:Reference Standards; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
chemical_list: D000143:Acids; D006020:Glycopeptides; D006023:Glycoproteins; D000509:alpha-Fetoproteins; D014357:Trypsin; D019158:N-Acetylneuraminic Acid