Immunoglobulin G Glycopeptide Profiling by Matrix-Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

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Abstract

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.

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