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.
Highlights
Glycoprotein structure determination and quantification by MS requires efficient isolation of glycopeptides from a proteolytic digest of complex protein mixtures
Glycoproteins Using Acids as Ion-pairing Reagents—A tryptic digest of an ribonuclease b (RNase B) and fetuin mixture was chosen to illustrate the use of IP-normal-phase LC (NPLC) for glycopeptide isolation (Fig. 1)
In this report we describe a new ion-pairing normal-phase LC (IP-NPLC) method for quantitatively isolating glycopeptides from complex tryptic digests using acids as ion-pairing reagents and polyhydroxyethyl A stationary phase
Summary
Materials and Reagents—Bovine RNase B, bovine fetuin, dithiothreitol, iodoacetamide, and the ion-pairing reagents were acquired from Sigma. MS Analysis of Tryptic Peptide Mixtures from Complex Protein Extracts—The tryptic digest of periplasmic protein extracts of the C. jejuni 11168 and the pglD mutant (40 g of each sample suspended in 7.5 l of 80% ACN ϩ 20% H2O ϩ 1% HCl ϩ 10 mM NH4HCO3) were repetitively subjected to IP-NPLC for off-line isolation of glycopeptides. The peak areas (signal/ noise Ն 3) from extracted ion chromatograms of label-free nanoRPLC-ESI-MS of the glycopeptides isolated by IP-NPLC were used for differential expression analysis of periplasmic glycoproteins between the wt and mutant strains. NanoRPLC-ESI-MS/MS analyses were performed with data-dependent analysis with a survey intensity threshold of 20 for the total digest and the glycopeptide and nonglycopeptide fractions of the periplasmic protein extract of the pglD mutant of C. jejuni 11168.
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