Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring

Abstract

Studies aimed toward glycan biomarker discovery have focused on glycan characterization by the global profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide new types of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. This new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profile and will thus be useful to determine if altered glycosylation profiles in serum/plasma are due to a change in protein glycosylation or a change in protein concentration. The remarkable sensitivity and selectivity of MRM enable the detection of low abundance IgG glycopeptides, even when IgG was digested directly in serum with no cleanup prior to the liquid chromatography. Our results show a low limit of detection of 60 amol and a wide dynamic range of 3 orders magnitude for IgG protein quantitation. The results show that IgG glycopeptides can be analyzed directly from serum (without enrichment) and yield more accurate abundances when normalized to the protein content. This report represents the most comprehensive study so far of the use of multiple reaction monitoring for the quantitation of glycoproteins and their glycosylation patterns in biofluids.

Figures

Figure 1

Representative Q-TOF tandem mass spectra of peptides and glycopeptides. (a) MS/MS spectrum of the peptide DTLMISR. (b) MS/MS spectrum of glycopeptide Hex5HexNAc4Fuc1Neu5Ac1-EEQYNSTYR from IgG1 (c) MS/MS spectrum of Hex3HexNAc4Fuc1- EEQFNSTFR from IgG2. ( ) N-acetylglucosamine ( ) mannose ( ) galactose (○) hexose ( ) fucose ( ) N-acetyl neuraminic acid

Figure 2

Total MRM chromatogram for IgG using UPLC-C18 chromatography. MRM chromatograms for 26 glycopeptides from (a) tryptic digested IgG standard and (b) tryptic digested pooled serum and (c) total MRM chromatogram for tryptic peptides from pooled serum. The MRM transitions are shown in Table 1. One MRM transition was monitored for each glycopeptide; two MRM transitions were monitored for each peptide.

Figure 3

Normalized abundance of IgG glycopeptides. (a) IgG1 glycopeptide MRM signal normalized to IgG1 peptide (FNWYVDGVEVHNAK) and ranked by the order of relative abundance. (b) IgG1 glycopeptide profiling using nano-chip-ESI-TOF-MS for the IgG standard. (c) IgG2 glycopeptide normalized to IgG2 peptides (CCVECPPCPAPPVAGPSVFLFPPKPK). (d) IgG3/4 glycopeptide normalized to IgG3 (WYVDGVEVHNAK) and IgG4 (TTPPVLDSDGSFFLYSR) peptides. The glycoform compositions are annotated above the histogram. For example, 4_4_1_1 corresponds to Hex4HexNAc4Fuc1Neu5Ac1.

Figure 4

Normalized (red squares) and unnormalized (blue bars) glycopeptide abundances of 4 IgG glycopeptides monitored in the sera of individuals. Normalization was performed using equation 2. (a) IgG1 glycopeptide, Hex4HexNAc4Fuc1-EEQYNSTYR (b) IgG1 glycopeptide, Hex3HexNAc4Fuc1-EEQYNSTYR (c) IgG2 glycopeptide, Hex4HexNAc4Fuc1-EEQFNSTFR (d) IgG2 glycopeptide, Hex3HexNAc4Fuc1-EEQFNSTFR. For each glycoform, the normalized trends in abundances do not directly match the unnormalized ones.