Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer

Abstract

Protein glycosylation fingerprints are widely recognized as potential markers for disease states, and indeed differential glycosylation has been identified in multiple types of autoimmune diseases and several types of cancer. However, releasing the glycans leave the glycoproteins unknown; therefore, there exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. In this study, a targeted multiple reaction monitoring (MRM)-based method for the protein- and site-specific quantitation involving serum proteins immunoglobulins A, G and M, alpha-1-antitrypsin, transferrin, alpha-2-macroglobulin, haptoglobin, alpha-1-acid glycoprotein and complement C3 was developed. The method is based on tryptic digestion of serum glycoproteins, followed by immediate reverse phase UPLC-QQQ-MS analysis of glycopeptides. To quantitate protein glycosylation independent of the protein serum concentration, a nonglycosylated peptide was also monitored. Using this strategy, 178 glycopeptides and 18 peptides from serum glycoproteins are analyzed with good repeatability (interday CVs of 3.65-21-92%) in a single 17 min run. To assess the potential of the method, protein glycosylation was analyzed in serum samples from ovarian cancer patients and controls. A training set consisting of 40 cases and 40 controls was analyzed, and differential analyses were performed to identify aberrant glycopeptide levels. All findings were validated in an independent test set (n = 44 cases and n = 44 controls). In addition to the differential glycosylation on the immunoglobulins, which was reported previously, aberrant glycosylation was also observed on each of the glycoproteins, which could be corroborated in the test set. This report shows the development of a method for targeted protein- and site-specific glycosylation analysis and the potential of such methods in biomarker development.

Keywords: LC-MRM-MS; biomarker discovery; epithelial ovarian cancer; glycopeptides; protein glycosylation; protein quantitation; serum.

Figure 1.

10% SDS PAGE of isolated IgG and IgA from Ascites 1 (asc1) and Ascites 412 (asc412) stained for protein. HC = heavy chain; LC = light chain. Mol. wt markers (kDa) are on left side of gel. Fluid is the original ascites fluid

Figure 2.

N-glycans identified in IgA and IgG from OC ascites fluid. A) Top 15 N-glycans from IgA isolated from asc1 and asc412 fluids, organized by abundance; B) Top 10 most abundance N-glycans in IgG isolated from asc1 and asc412 fluids. Blue bars represent N-glycans from asc1; red bars represent those from asc412. Annotated N-glycan structures are shown with mannose (green circle); galactose (yellow circle); GlcNac (N-acetyl glucosamine)(blue square); fucose (red triangle) and sialic acid (S)(purple diamond).

Figure 3.

N-glycans from site specific analyses of IgG and IgA isolated from asc1 and asc412 ascites fluids. Peptides were digested by pronase followed by site specific glycan analysis using nLC Chip/qTOF MS. A) N-glycans identified in site N297 in IgG heavy chain from asc1 and asc412. Red box identifies non-fucosylated biantennary N-glycans in asc412; B) N-glycans identified in specific N-glycan sites in IgA1 and IgA2 isolated from OC asc1 and asc412. N-glycan sites are indicated in the sequence by red numbers for N144, N340 in IgA1 and N47, N92, N131, N205 and N327 in IgA2 for each ascites fluid. Structures of N-glycans identified by tandem MS/MS and based on composition are indicated below each glycan site in IgG, IgA1 and IgA2 from asc1 and asc412.

Figure 4.

Tandem MS/MS analysis of N-glycans produced by pronase digestion. A) MS/MS analysis of N-glycan H3N5F0S0 attached to the same IgA1 site (N131/144) in both ascites fluids (asc1 and asc412); B) MS/MS analysis of a different N-glycan H5N4F0S1 attached to the same site; C) MS/MS analysis of N-glycan H5N4F1S1 attached to N340 in IgA1 in both ascites fluids; D) MS/MS of N-glycan H5N4F1S2 attached to N340 in IgA1 in both ascites fluids; E) MS/MS of N-glycan H4N4F2S0 attached to glycan site N205 in asc1 IgA2, but not detected in asc412 IgA2.