Glycopeptide Compositional Analysis

Glycopeptide compositional analysis was performed from m/z features using in-house written SysBioWare software (Vakhrushev et al., 2009 (link)). For m/z feature recognition from full MS scans Minora Feature Detector Node of the Proteome discoverer 2.3 (ThermoFisher Scientific) was used. A list of precursor ions (m/z, charge and retention time) was imported as ASCII data into SysBioWare and compositional assignment within 5 ppm mass tolerance was performed. The main building blocks used for the compositional analysis were: NeuAc, Hex, HexNAc, dHex and phosphate. The most prominent peptides corresponding to each potential glycosite were determined experimentally by comparing the yield of deamidated peptides before and after PNGase F treatment. The peptide sequence was determined by HCD MS/MS and the abundance level was calculated from PD 2.3. For N-glycopeptide compositional analysis the corresponding peptides were also added as building blocks.
A list of potential glycan and glycopeptides for each glycosite was generated and the top 10–15 of the most abundant candidates were selected for targeted MS/MS analysis to confirm the proposed structure. Each targeted MS/MS spectrum was subjected to manual interpretation. Since the same N-glycan composition may represent various isobaric structures, the final glycan structures were proposed according to literature data, predicted enzyme functions of the targeted genes, along with information in MS/MS fragments.