Extended Data Fig. 5: Glycopeptides carrying NeuGc and multi-Fuc signatures are undetectable or rarely detected in the human serum sample investigated in this study.

a. Extracted ion chromatograms (XICs) were performed at the MS/MS levels for well-established diagnostic oxonium ions, including fragment ions reporting on i) HexNAc, ii) NeuAc, and iii) NeuGc. While abundant diagnostic ions as expected were observed for HexNAc and NeuAc, practically no diagnostic ions were observed for NeuGc glycopeptides. The XIC traces have been plotted on the same absolute intensity scale. All fragmentation modes (HCD, EThcD and CID) were considered for this XIC analysis. Only data from File B reported on by all teams were plotted in this figure; File A showed similar patterns (data not shown). b. Example of an HCD-MS/MS spectrum of a NeuAc-containing sialoglycopeptide correctly and incorrectly annotated by teams. Most teams correctly identified that this scan corresponds to a NeuAc glycopeptide as demonstrated by the presence of diagnostic oxonium and B ions for NeuAc, while two teams incorrectly identified the spectrum as a NeuGc-containing glycopeptide despite the absence of diagnostic oxonium and B ions for NeuGc (see insert) and one team incorrectly identified the spectrum as a NeuAc and Fuc containing glycopeptide due to the misidentification of Met oxidation. c. XICs were performed at the MS/MS level for well-established diagnostic B ions reporting on different antenna features, including i) sialyl LacNAc, ii) sialyl Lewis x/a, and iii) Lewis x/a. While abundant diagnostic ions as expected were observed for sialyl LacNAc, only very few diagnostic ions were observed for antennary fucosylation features (sialyl Lewis x/a and Lewis x/a). iv) Few diagnostic ions for antenna fucosylation could be observed at very low abundance, which indicated that antenna fucosylation (and thus by extension multi-fucosylated glycopeptides) are present but are rarely detected in the studied serum sample. The XIC traces have been plotted on the same absolute intensity scale. All fragmentation modes (HCD, EThcD and CID) were considered for this XIC analysis. Only data from File B reported on by all teams were plotted in this figure; File A showed similar patterns (data not shown). d. Example of an HCD-MS/MS spectrum of a multi-Fuc-containing glycopeptide correctly and incorrectly annotated by teams. Most teams correctly identified that this scan corresponds to a multi-Fuc sialoglycopeptide as indicated by the presence of diagnostic B ions for Lewis x/a (see insert, broken lines) and NeuAc oxonium ions as well as core fucosylated Y1 and Y2 ions, while one team incorrectly identified the spectrum as a tetra-fucosylated asialylated glycopeptide. Note that some teams (for example team 17) reported on several different 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) supported that this spectrum corresponds to a difucosylated glycopeptide carrying a single NeuAc. e. Example of an HCD-MS/MS spectrum of a NeuAc-containing glycopeptide correctly and incorrectly annotated by teams. Three teams correctly identified that this scan corresponds to a disialylated (NeuAc) afucosylated glycopeptide as indicated by the presence of diagnostic oxonium and B ions for NeuAc, while three teams incorrectly identified the spectrum as a multi-Fuc sialoglycopeptide despite the lack of diagnostic ions for core fucosylated Y1 ions, and sialyl Lewis x/a or Lewis x/a. The monoisotopic precursor ion profile (see insert, full lines) supported that this spectrum corresponds to a disialylated NeuAc glycopeptide not carrying fucose.