The generated raw files containing the acquired mass spectra were converted to mzXML files using the msconvert utility in the Trans-Proteomic Pipeline software. The “centroid all scans” option was selected. The mzXML file corresponding to each of the tryptic global peptide runs was opened in MATLAB. The MS/MS spectra of the glycopeptides were distinguished from peptide MS/MS based on the presence of oxonium ions. These ions belong to glycan free monosaccharides or disaccharides that were fragmented during the tandem mass spectrometry analysis. In this step, the MS/MS spectra including at least two of the oxonium ions with the masses of 138 (internal fragment of HexNAc), 145 (Hex–H2O), 163 (Hex), 168 (HexNAc–2H2O), 186 (HexNAc–H2O), 204 (HexNAc), 325 (Hex2), 366 (HexHexNAc), 274 (Neu5Ac–H2O), or 292 (Neu5Ac) were isolated as oxoniumion-containing spectra. For the spectra with more than 100 peaks, oxonium ions were searched in the top 10% of the mass spectral peaks within a 10 ppm window.
Protocol detail
Mass Spectrometric Analysis of Glycopeptides
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Disaccharides
Glycan
Glycopeptides
Ions
Mass spectra
Monosaccharides
Ms ms
Oxonium
Peptide
Scans
Tryptic
Corresponding Organization :
Other organizations : Johns Hopkins Medicine, Johns Hopkins University
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Variable analysis
independent variables
- Selection of the 'centroid all scans' option in the msconvert utility
- Distinction of glycopeptide MS/MS spectra from peptide MS/MS based on the presence of oxonium ions
- Identification of glycopeptide MS/MS spectra containing at least two of the specified oxonium ions
- The mass spectra were acquired and converted to mzXML files
- The mzXML files corresponding to the tryptic global peptide runs were opened in MATLAB
- No positive or negative controls were explicitly mentioned in the input protocol.
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