Semi-Automated Identification of N-Glycopeptides by Hydrophilic Interaction Chromatography, nano-Reverse-Phase LC–MS/MS, and Glycan Database Search

Abstract

Glycoproteins fulfill many indispensable biological functions and changes in protein glycosylation have been observed in various diseases. Improved analytical methods are needed to allow a complete characterization of this complex and common posttranslational modification. In this study, we present a workflow for the analysis of the microheterogeneity of N-glycoproteins which couples hydrophilic interaction and nano-reverse-phase C18 chromatography to tandem QTOF mass spectrometric analysis. A glycan database search program, GlycoPeptideSearch, was developed to match N-glycopeptide MS/MS spectra with the glycopeptides comprised of a glycan drawn from the GlycomeDB glycan structure database and a peptide from a user-specified set of potentially glycosylated peptides. Application of the workflow to human haptoglobin and hemopexin, two microheterogeneous N-glycoproteins, identified a total of 57 distinct site-specific glycoforms in the case of haptoglobin and 14 site-specific glycoforms of hemopexin. Using glycan oxonium ions, peptide-characteristic glycopeptide fragment ions, and by collapsing topologically redundant glycans, the search software was able to make unique N-glycopeptide assignments for 51% of assigned spectra, with the remaining assignments primarily representing isobaric topological rearrangements. The optimized workflow, coupled with GlycoPeptideSearch, is expected to make high-throughput semi-automated glycopeptide identification feasible for a wide range of users.

Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma

“…The two-dimensional HILIC and nano-reverse phase C18 chromatography was coupled to a QTOF mass spectrometer as described previously (29). This configuration achieves an efficient enrichment of glycopeptides and sensitive detection of the glycoforms of the individual peptides.…”

“…Here, we describe changes in the site-specific glycoforms of Hp associated with liver cirrhosis and HCC. We expand our recently reported LC-MS/MS analysis of Hp (29) by exoglycosidase treatment of glycopeptides, immunoblotting, and MS/MS analysis of detached permethylated glycans in the context of liver disease.…”

“…Protein Digestion and Exoglycosidase Treatment-Hp was digested as described previously (29). Briefly, 2.5 g of isolated Hp was resuspended in 20 l of 50 mM NH 4 HCO 3 at pH 7.8 (Sigma-Aldrich) with 0.05% RapiGest (Waters, Milford, MA), reduced with 5 mM DTT and alkylated with 15 mM iodoacetamide (Sigma-Aldrich).…”

“…Separation of Glycopeptides by HILIC and Nano-reverse Phase C18 Chromatography-Ten micrograms of a tryptic digest of Hp isolated from all samples were resuspended in 20 l of 50% ACN in water (Fisher Scientific, Fair Lawn, NJ) and injected on a HILIC XBridge column (5 m, 2.1 ϫ 100 mm) (Waters) as described previously (29). Briefly, the column was equilibrated in 90% of solvent B (0.01% TFA in acetonitrile (Fisher Scientific)), and separation was achieved using a 35-min linear gradient of 90 to 40% solvent B at 40°C (solvent A, 0.01% TFA in water; solvent B, 0.01% TFA in acetonitrile).…”

Glycoprotein Disease Markers and Single Protein-omics

“…Mutations of the NX(S/T) sequon occur frequently in disease context (30). Nonsynonymous genomic variants create or abolish NX(S/T) sites on several thousand human proteins and can be associated with cancer risk (31). Relocation of GalNAc transferases to the ER in cancer cells is associated with increased multiplicity of O-glycosylation (14), and Liu et al (32) reported changes in site occupancy at several N-glycosylation sites in patients with ovarian cancer.…”