Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC–MS–MRM in liver disease

Abstract

Aberrant glycosylation of IgGs has been linked to human diseases, including liver disease. In this study, we have quantified plasma immunoglobulins in cirrhosis (CIR) and hepatocellular carcinoma (HCC) and employed a novel LC-MS-MRM assay to quantify glycoforms of IgG subclasses 1-4. Glycan oxonium ions and peptide-GlcNAc fragment ions were utilized to quantify the IgG glycoforms purified by affinity chromatography with normalization to the unique peptide for each IgG subclass. Our results indicate that HCC patients have increased circulating IgG1, IgG3, IgA1, and IgM compared to healthy controls; comparison of HCC and CIR patients shows that HCC patients have significantly higher concentration of IgG1 and IgM but lower concentration of IgG2. Increase in galactose-deficient core fucosylated glycoforms was consistently observed in CIR and HCC patients. The FA2G0 and FA2BG0 glycoforms increase approximately 2-fold in all IgG subclasses accompanied by a decrease in the FA2G2 glycoform. Fucosylation changes are less pronounced but we have detected increased degree of fucosylation in the IgG1 and IgG3 glycoforms. In conclusion, we have optimized a sensitive and selective LC-MS-MRM method for quantification of immunoglobulin subclasses and their site specific glycoforms, demonstrating that both quantities and glycoforms of immunoglobulins change significantly in liver disease progression to HCC.

Data Independent Analysis of IgG Glycoforms in Samples of Unfractionated Human Plasma

“…40,41 Figure 1A shows the spectrum of the G2F glycoform of IgG1 obtained under rolling CE conditions optimized for the charge state and precursor mass of the peptide. Under these conditions, intense oxonium B-ions are accompanied by low intensity Y1 fragments with/out fucose.…”

“…13,16 This enrichment step enables the use of the methods developed for the analysis of therapeutic IgG provided that sufficient amounts of representative IgG glycoforms are accessible. 14,17–19 …”

“…14,19 The available MS methods are based most frequently on the quantification of precursor ion intensities 13,20 because the signal, especially in the case of isolated IgG, is typically sufficient for adequate coverage of the glycoform precursors. 14,21 Several groups reported also MRM quantification of oxonium ions generated by collision induced dissociation (CID) of glycopeptides as reviewed in ref 15.…”

“…These common glycan fragments ( m / z 138, 204, 366) have high sensitivity but low specificity because they are generated from virtually all N-glycopeptides and have interferences from some peptide fragments as well. 22,23 Nonetheless, these B-ions, in the Domon/Costello nomenclature, 24 were used for quantification of IgG glycoforms in both samples of isolated IgG 19,25 and in complex biological samples. 26 Abundance of IgG in biological samples is sufficient to allow label free quantification of at least the more abundant glycoforms using the MRM of B-ions; however, coverage of the low-abundance glycoforms is lower in the complex biological matrix.…”

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Several applications of multiple reaction monitoring (MRM)-based glycopeptide analysis from a variety of biological systems and diseases were lately published including the use of MRM-based serum glycoproteomics to study esophagus diseases (140), liver disease (141), and the immunoglobulin subclasses (134,142,143). Among other technical challenges unique to MRM-based glycopeptide detection and quantitation, one of the most important considerations is the selection of the precursor-product transitions being monitored.…”