Glycopeptides (1.5 µg) were loaded on a 50 cm ES803 column (Thermo Fisher Scientific). Peptides were separated using a 2-hour gradient, at 250nL/min flow, using the EasyLC1000 nano-liquid-chromatography system (Thermo Fisher Scientific). The chromatography system was coupled to an Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific) and MS/MS data were acquired in a data dependent mode. The Orbitrap mass detector was used for the MS/MS acquisition, and the maximum injection time was set to 100 milliseconds for enhanced sensitivity.
The acquired raw data were analyzed by the Max Quant software (version 1.6.1.0), using the complete human proteome (version 2016.07.13 containing 42,041 sequences) downloaded from UniProt. Searches were performed with a maximum of two missed cleavages. Carbamido-methylation of cysteines was specified as fixed modification, while oxidation of methionine and deamidation of asparagine to aspartic acid (0.98Da mass shift) were specified as variable modifications. False discovery of peptides was controlled using a target-decoy approach based on reversed sequences, and the false discovery rate was defined as 1% at site, peptide, and protein levels.
The acquired raw data were analyzed by the Max Quant software (version 1.6.1.0), using the complete human proteome (version 2016.07.13 containing 42,041 sequences) downloaded from UniProt. Searches were performed with a maximum of two missed cleavages. Carbamido-methylation of cysteines was specified as fixed modification, while oxidation of methionine and deamidation of asparagine to aspartic acid (0.98Da mass shift) were specified as variable modifications. False discovery of peptides was controlled using a target-decoy approach based on reversed sequences, and the false discovery rate was defined as 1% at site, peptide, and protein levels.