Extended Data Fig. 6: Examples of (in)correctly annotated N-glycopeptides.

a. HCD-MS/MS fragment spectrum of a ‘consensus’ NeuAc-containing sialoglycopeptide correctly annotated by all 16 teams (teams 1, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21) reporting on this particular scan number. Manual annotation confirmed that this spectrum indeed corresponds to the indicated NeuAc-containing N-glycopeptide from human alpha-2-HS-glycoprotein (UniProtKB, P02765) as demonstrated by the presence of diagnostic oxonium and B ions for NeuAc and extensive b- and y-ion peptide backbone fragmentation. Further, the monoisotopic precursor ion profile (see insert) supported the annotation of this spectrum. b. HCD-MS/MS spectrum of a NeuAc-containing core-fucosylated glycopeptide that was incorrectly annotated by several teams. While four teams (teams 10, 17, 20, 21) correctly identified that this spectrum corresponds to an N-glycopeptide from human immunoglobulin heavy constant mu (P01871) carrying a single NeuAc and Fuc as indicated by the presence of diagnostic oxonium and B ions for NeuAc (see insert, broken lines), y-ions confirming Met oxidation and Cys carbamidomethylation, and correct monoisotopic precursor ion profile, four incorrect glycan structures were reported by other teams as indicated. The structural differences between the incorrectly and correctly assigned glycans have been indicated in attempts to rationalize the misidentification. All teams (except for team 1, who reported a different peptide from a different source protein with an incorrect precursor m/z, data not shown) identified the correct peptide sequence, although the Met oxidation and Cys carbamidomethylation were features that frequently led to incorrect glycopeptide identification. Some teams (for example team 21) reported on several glycopeptides from the same scan, likely due to conflicting output data from multiple searches of the same data. The monoisotopic precursor ion profile (see insert, full lines) and the subsequent EThcD-MS/MS scan (scan #8026, data not shown) supported that this spectrum, in fact, corresponds to the indicated N-glycopeptide carrying Met oxidation and Cys carbamidomethylation as well as an N-glycan displaying a composition corresponding to a complex N-glycan structure with a single NeuAc and Fuc.