Comparative Glycomics of Immunoglobulin A and G From Saliva and Plasma Reveals Biomarker Potential

Abstract

The N-glycosylation of immunoglobulin (Ig) G, the major antibody in the circulation of human adults, is well known for its influence on antibody effector functions and its alterations with various diseases. In contrast, knowledge on the role of glycans attached to IgA, which is a key immune defense agent in secretions, is very scarce. In this study we aimed to characterize the glycosylation of salivary (secretory) IgA, including the IgA joining chain (JC), and secretory component (SC) and to compare IgA and IgG glycosylation between human plasma and saliva samples to gain a first insight into oral cavity-specific antibody glycosylation. Plasma and whole saliva were collected from 19 healthy volunteers within a 2-h time window. IgG and IgA were affinity-purified from the two biofluids, followed by tryptic digestion and nanoLC-ESI-QTOF-MS(/MS) analysis. Saliva-derived IgG exhibited a slightly lower galactosylation and sialylation as compared to plasma-derived IgG. Glycosylation of IgA1, IgA2, and the JC showed substantial differences between the biofluids, with salivary proteins exhibiting a higher bisection, and lower galactosylation and sialylation as compared to plasma-derived IgA and JC. Additionally, all seven N-glycosylation sites, characterized on the SC of secretory IgA in saliva, carried highly fucosylated and fully galactosylated diantennary N-glycans. This study lays the basis for future research into the functional role of salivary Ig glycosylation as well as its biomarker potential.

Quantitative determination of human IgA subclasses and their Fc‐glycosylation patterns in plasma by using a peptide analogue internal standard and ultra‐high‐performance liquid chromatography/triple quadrupole mass spectrometry

“…Compared with previous studies of IgA analysis, we provided detailed optimization steps of using cost-effective peptide M beads and the developed method was carefully validated in terms of precision, accuracy, linearity, matrix effects and stability. [14][15][16][17] We also applied the method to 30 clinical samples, and the quantification results of total IgA from the current method showed good correlation with the results from the commercial ELISA kit. In addition, this UHPLC/MS/MS method also provides subclass information and glycopeptide responses.…”

“…Surrogate peptides and Fc‐glycopeptides of IgA1 and IgA2 were selected based on information from the literature . The MRM parameters used for detection of IgA1 (TPLTATLSK) and IgA2 (DASGATFTWTPSSGK) surrogate peptides were first predicted by Skyline software and determined by the experimental results where transitions and parameters with higher responses were selected.…”

“…The method achieved absolute quantification of IgA subclasses and relative quantification of IgA glycopeptides. Compared with previous studies of IgA analysis, we provided detailed optimization steps of using cost‐effective peptide M beads and the developed method was carefully validated in terms of precision, accuracy, linearity, matrix effects and stability . We also applied the method to 30 clinical samples, and the quantification results of total IgA from the current method showed good correlation with the results from the commercial ELISA kit.…”

“…Mass spectrometry is a selective and sensitive detection method that can be applied to clinical biofluids and tissues to identify minor components from a complicated matrix. A few studies have developed liquid chromatography/tandem mass spectrometry (LC/MS) methods using the multiple reaction monitoring (MRM) mode to determine IgA Fc N‐glycosylation in biofluids, such as human milk, saliva and plasma . In these studies, IgG and IgM‐glycopeptides were also included as target analytes.…”

“…A few studies have developed liquid chromatography/tandem mass spectrometry (LC/MS) methods using the multiple reaction monitoring (MRM) mode to determine IgA Fc N-glycosylation in biofluids, such as human milk, saliva and plasma. [14][15][16][17] In these studies, IgG and IgM-glycopeptides were also included as target analytes. IgA subclasses and IgA Fc Nglycosylation are also important to investigate.…”