Fig. 3. Outline of identified sugar components and observed oligosaccharide profiles for the Ca. Kuenenia stuttgartiensis enrichment.

a The large pie chart outlines the proportions of the carbohydrate fragments identified in the Ca. Kuenenia stuttgartiensis enrichment proteome using the MS2 mass binning approach. The lower charts depict the proportions of spectra containing carbohydrate related signals (oxonium ions) in the sequencing spectra, and the frequency of individual glycoforms across all spectra. b The graphs outline the furthermore established oligosaccharide chains using parent ion offset binning of fragmentation spectra containing the identified carbohydrate fragments (graphs labeled with 204, 275, 289, and 175 m/z). Thereby, oligosaccharide chains appear in histograms as repeatedly occurring mass deltas. The same parent ion offset approach applied to the complete, non-carbohydrate-filtered dataset, does not reveal any identifiable systematically reoccurring mass deltas (bottom graph). This revealed two completely unrelated oligosaccharide chains. The fragments 204, 261, 275, and 289 m/z belong to variations of a complex type oligosaccharide with a HexNAc core structure (X-type). The fragment 175 (and 193 m/z) retrieved a second, fully unrelated heptose type oligosaccharide chain (O-type). Squares represent HexNAcs (methylations are depicted by a dot), diamonds represent NulO variants, hexagons represent heptoses, triangles are deoxyhexoses, and doted triangles are dimethyl-deoxyhexoses. Moreover, due to the predominant fragmentation of the oligosaccharide chains, nearly the complete sequence of the oligosaccharide can be derived. c The histograms outline the (intensity normalized) low mass bins of fragmentation spectra where the complex type oligosaccharide (upper graph) or the oligo-heptosidic chains (O-type, lower graph) were identified. The thereby-observed sugar fragments correlate with the proposed composition of the individual carbohydrate chains (e.g., 204/261 m/z for complex, or 175/193 m/z for oligo-heptosidic). d The histogram shows binning of mass deltas into very small bin sizes to establish the oligosaccharide compositions at very high mass accuracy (<7.5 ppm).