Extended Data Fig. 8: Data underpinning the synthetic N-glycopeptide performance test (N1).

a. MS/MS spectra corresponding to the non-adducted synthetic N-glycopeptide (EVFVHPNYSK, Hex₅HexNAc₄NeuAc₂, UniProtKB, P04070) in charge state 3⁺ and 4⁺ (9 top spectra) and the K⁺-adducted synthetic N-glycopeptide in charge state 5⁺ (three bottom spectra) arising from the four fragmentation modes (HCD-, ETciD-, EThcD- and CID-MS/MS) used to generate File A and B. Green asterisks: Oxonium ions and non-reducing end glycan fragments (B-ions). Blue asterisks: Y-ion series (peptide conjugated with glycan fragment). Red asterisks: Peptide backbone b-/y-/c-/z-ions. Black asterisks: Unfragmented peptide without glycan, unfragmented precursor (peptide with glycan) and charge-reduced precursor. b. Overview of the 12 MS/MS spectra of the synthetic N-glycopeptide (from panel a) that were either correctly identified (green), incorrectly identified (red), or not reported by each team (white). Spectra arising from fragmentation mode(s) not included in the search strategy chosen by each team were not included in the assessment (indicated in grey). c. Structure of the synthetic N-glycopeptide spiked into the human serum sample. d. Performance scores arising from the test determined for each team based on the sensitivity and specificity of the identification of the 12 MS/MS spectra corresponding to the synthetic N-glycopeptide.