Fc Glycopeptide Identification by LC-MS

Glycopeptides were separated with an Ultimate 3000 RSLCnano high-performance liquid chromatography system (Thermo Scientific, Waltham, MA) equipped with an Acclaim PepMap 100 trap column (100 μm × 20 mm, 5 μm particle size; Thermo Scientific) and an Acclaim PepMap RSLC C18 nano-column (75 μm × 150 mm, 2 μm particle size) analytical column. Five hundred nL of total IgG and two hundred nL of HLA-A2-specific IgG was injected and separated with a gradient from 97% solvent A (0.1% formic acid in water) and 3% solvent B (95% ACN) down to 27% solvent B, at a flowrate of 700 nL/min over 15 minutes. The LC-MS system was hyphenated to a maXis HD quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Billerica, MA) via an electrospray ionization interface, which was equipped with a CaptiveSpray nanoBooster using ACN-enriched nitrogen gas (at 0.2 bar pressure and a dry gas flow rate of 3 L/min). A frequency of 1 Hz was used for recording the spectra in the m/z range of 550–1800 in positive ion polarity mode. The transfer time was set to 130 μs, the pre-pulse storage time to 10 μs, while the collision energy was set to 5 eV.¹¹³ (link) This method allowed unambiguous identification of IgG Fc glycopeptides in a subclass specific manner based on accurate mass (MS1) and specific migration positions in liquid chromatography.