Immunoglobulin G glycopeptide profiling by matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry.
Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 microL of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples.
Selman
Maurice H J
MH
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
McDonnell
Liam A
LA
Palmblad
Magnus
M
Ruhaak
L Renee
LR
Deelder
André M
AM
Wuhrer
Manfred
M
D000328:Adult; D000368:Aged; D015331:Cohort Studies; D005260:Female; D005583:Fourier Analysis; D006020:Glycopeptides / Q000032:analysis* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D050440:Immunoglobulin Subunits; D008297:Male; D008875:Middle Aged; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D013205:Staphylococcal Protein A / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006020:Glycopeptides; D007074:Immunoglobulin G; D050440:Immunoglobulin Subunits; D013205:Staphylococcal Protein A; D014357:Trypsin