Abstract
Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.
Highlights
Protein glycosylation is an important biological process that refers to the enzymatic attachment of carbohydrates to proteins
Recent studies have shown that different Immunoglobulin G (IgG) subclasses tend to have distinct fragment crystallizable (Fc) region N-glycosylation[19,20], which might contribute to their functionality
These aliquots were pooled at different steps of the sample preparation procedure, designated as conditions A–E (18–19 replicates per condition), to get rid of the technical variation introduced by the previous steps (Fig. 1)
Summary
Protein glycosylation is an important biological process that refers to the enzymatic attachment of carbohydrates to proteins. A few recent publications described Fc-linked IgG N-glycans in common laboratory mouse strains[19,30] and effects of immunization on antigen-specific IgG N-glycosylation profile[31]. In this study we assessed the reliability of the high-throughput nano-LC-ESI-MS method for quantification of mouse IgG subclass specific Fc N-linked glycopeptides.
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