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Molecular & Cellular Proteomics 8:2170-2185, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification and Quantification of Glycoproteins Using Ion-Pairing Normal-phase Liquid Chromatography and Mass Spectrometry^*,

Wen Ding^,, Harald Nothaft^,¶, Christine M. Szymanski^,¶ and John Kelly

From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6 and
¶Department of Biological Sciences, Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada

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.

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