HBP N-Glycoproteomic data analysis workflow. (IMAGE)
Caption
The intact N-glycopeptides, present in 24 glycopeptide enriched fractions (12× elution 1 + 12× elution 2) were analyzed by LC-MS/MS using both fragmentation energies, HCD.step and HCD.low. A1 and A2: The spectra file acquired at each fragmentation regime was searched for N-glycopeptides using Byonic software. B1 and B2: All individual searches corresponding to the same fragmentation energy were combined using Byologic. After this step, two large N-glycopeptide identification lists were produced: HCD.step-HBP list and HCD.low-HBP list. C: The N-glycopeptides contained in the HCD.step-HBP list were manually validated to confirm the peptide and N-glycan composition assigned by the software. D: The N-glycopeptide-identifications confirmed were imported into the HCD.low-HBP list to substitute corresponding precursor ions potentially incorrectly identified, using their mass and retention time. E: Features annotated in the HCD.step-HBP list were transferred to the HCD.low-HBP list and an additional revision for N-glycan structural evidence is conducted in the HCD.low–HPB list. F: A second Byonic search focused on the new features identified was triggered. G: Finally, a second HCD.step-HBP list containing corrected N-glycopeptide identifications was generated using Byologic. Image created with BioRender.com.
Credit
Frania J. Zuniga-Banuelos et al.
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