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 nonglycopeptides 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 nonglycopeptides relative to glycopeptides in IP-NPLC is predictable by their hydrophobicity values calculated using the Wimley-White water/octanol hydrophobicity scale. Olinked 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 wildtype 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. Molecular & Cellular Proteomics 8:2170 -2185, 2009.Protein glycosylation is a biologically significant and complex post-translational modification, involved in cell-cell and receptor-ligand interactions (1-4). In fact, clinical biomarkers and therapeutic targets are often glycoproteins (5-9). Comprehensive glycoprotein characterization, involving glycosylation site identification, glycan structure determination, site occupancy, and glycan isoform distribution, is a technical challenge particularly for quantitative profiling of complex protein mixtures (10 -13). Both N-and O-glycans are structurally heterogeneous (i.e. a single site may have different glycans attached or be only partially occupied). Therefore, the MS 1 signals from glycopeptides originating from a glycoprotein are often weaker than from non-glycopeptides. In addition, the ionization efficiency of glycopeptides is low compared with that of non-glycopeptides and is often suppressed in the presence of non-glycopeptides (11-13). When the MS signals of glycopeptides are relatively high in simple protein digests then diagnostic sugar oxonium ion fragments produced by, for example, front-end collisional activation can be used to detect them. However, when peptides and glycopeptides coelute, parent ion scanning is required to selectively detect the glycopeptides (14). This can be problematic in terms of sensitivity, especially for detecting glycopep...

Modification of the Campylobacter jejuni N-Linked Glycan by EptC Protein-mediated Addition of Phosphoethanolamine

“…Manual interrogation of MS/MS spectra concentrated on high quality scans, with clear glycan fragment ions but no positive assignment by MASCOT. These spectra contained no evidence for the monoacetylated Bac linked to Asn previously observed in the laboratory passaged NCTC 11168 strain (32). Furthermore, we found no examples of truncated or elongated N-glycans modified by the addition or subtraction of GalNAc residues.…”

“…Analysis of fOS (30), N-linked glycan (22,30), and pathway intermediates (e.g. lipid-bound glycan (31)) suggest that the heptasaccharide is the sole N-glycan formed, although enzymes within the pgl biosynthetic pathway appear to have broader specificity than the substrates used to construct the canonical glycan (25,(32)(33). Monoacetylated Bac in the laboratory passaged NCTC 11168 strain (32) remains the only glycan variant identified todate.…”

“…lipid-bound glycan (31)) suggest that the heptasaccharide is the sole N-glycan formed, although enzymes within the pgl biosynthetic pathway appear to have broader specificity than the substrates used to construct the canonical glycan (25,(32)(33). Monoacetylated Bac in the laboratory passaged NCTC 11168 strain (32) remains the only glycan variant identified todate. As passaged C. jejuni isolates differ in the manifestation of phenotypes associated with motility, morphology, and virulence (34,35), it is unclear if the utilization of monoacetylated Bac is an authentic process common to all C. jejuni.…”

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

“…Because of this challenge a deeper understanding of the glycan diversity and substrates of glycosylation has been largely unachievable for the majority of known bacterial glycosylation systems. The recent implementation of selective glycopeptide enrichment methods (26,27) and the use of multiple fragmentation approaches (28,29) has facilitated identification of an increasing number of glycosylation substrates independent of prior knowledge of the glycan structure (30 -33). These developments have facilitated the undertaking of comparative glycosylation studies, revealing glycosylation is widespread in diverse genera and far more diverse then initially thought.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…62 Polar glycopeptides are retained on the HILIC phase, while the hydrophobic peptides are washed off with high organic concentrations in the mobile phase, which allows the separation of glycosylated and non-glycosylated peptides. 119,120 Retained glycopeptides can then be eluted by increasing the water content of the mobile phase. Recently, tryptic glycopeptides have been finely separated on a 1.7-μm amide column, with elution in the order of non-glycosylated, O-glycosylated and N-glycosylated glycopeptides.…”