Byonic version 2.7

Eluted glycopeptides were dried again and re-suspended in 0.1% formic acid prior to mass spectrometry analysis. An aliquot of glycopeptides was analyzed by LC-MS with an Easy-nLC 1200 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) using higher energy collisional dissociation (HCD) fragmentation. Peptides were separated using an EasySpray PepMap RSLC C18 column (75 μm x 75 cm) with a 275 minute linear gradient consisting of 0%-32% acetonitrile in 0.1% formic acid over 240 minutes followed by 35 minutes of 80% acetonitrile in 0.1% formic acid. The flow rate was set to 200 nL/min. The spray voltage was set to 2.8 kV and the temperature of the heated capillary was set to 275 °C. HCD collision energy was set to 50%, appropriate for fragmentation of glycopeptide ions. Glycopeptide fragmentation data were extracted from the raw file using Byonic™ (Version 2.7) and Byologic™ software (Version 2.3; Protein Metrics Inc.). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The relative abundance of each glycoform at each site was calculated using the extracted ion chromatograms for truepositive peptides.