Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum.
All four subclasses of human serum IgG contain a single N-glycosylation site in the constant region of their heavy chain, which is occupied by biantennary, largely core-fucosylated and partially truncated oligosaccharides, that may carry a bisecting N-acetylglucosamine and sialic acid residues. IgG glycosylation has been shown to be altered under various physiological and pathological circumstances. IgG N-glycan profiles vary with age, and galactosylation for example is enhanced during pregnancy. Several diseases including rheumatoid arthritis are associated with a reduction in galactosylation of the IgG N-glycans. Here, we describe a robust method for the isolation of IgG subclasses using protein A (binds IgG1, IgG2, and IgG4) and protein G (binds additionally IgG3) at the 96-well plate level, which is suitable for automation. Isolated IgGs were digested with trypsin, and obtained glycopeptides were analyzed by nano-LC-MS. Glycopeptides were characterized by CID as well as electron transfer dissociation (ETD). The method provided glycosylation profiles for IgG1, IgG2, IgG3, and IgG4 and revealed distinct differences in N-glycosylation between the four IgG subclasses. The changes in galactosylation associated with rheumatoid arthritis could readily be monitored. This method is suitable for the subclass-specific analysis of IgG glycosylation from clinical samples.
Wuhrer
Manfred
M
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. m.wuhrer@lumc.nl
Stam
Jord C
JC
van de Geijn
Fleur E
FE
Koeleman
Carolien A M
CA
Verrips
C Theo
CT
Dolhain
Radboud J E M
RJ
Hokke
Cornelis H
CH
Deelder
André M
AM
D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000145:classification / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry
D006020:Glycopeptides; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D014357:Trypsin