Fig. 1. Universal sialic acid large-scale survey approach. (A) outlines the three main NulO core structures known to date: Kdn (deaminoneuraminic acid), Neu (neuraminic acid) and P.L.A = Pse (pseudaminic acid), Leg (legionaminic acid) and Aci (acinetaminic acid). (B) demonstrates the potential diversity generated through (combinations) of (natural) diversifications of the three main NulO core compositions Kdn, Neu and P.L.A (Pse/Leg/Aci). The left graph shows a binned mass histogram (5 Da) of the theoretically possible chemical compositions between 380–520 Da. Already more than 25 different compositions are found within ±0.05 Da of Pse5Ac7Ac. The image on the right shows the mass defect binned for every calculated structure, which all fall within a very narrow window between 0.05–0.25 Da. The mass spectrometric mass defect is defined as the delta of monoisotopic mass and nominal mass. For combinatorial space and abbreviations see ESI-Table, sheet 8.‡ This filters for possible realistic structures during parent NulO annotation. (C) outlines the established large-scale survey pipeline starting from crude cell lysates, α-keto acid specific chemical labelling, small mass channel scanning (2.75 m/z windows, from 380–520 Da), and the automated structural filtering and evidence scoring pipeline to annotate parent NulOs. In summary, channel hits and compositions are discovered based on highly conserved quinoxaline-core fragments together with the diversification-independent carbon chain length and structural features.