FIGURE 1.

Orbitrap MS analysis of the TgSkp1 glycopeptide. RHΔΔ tachyzoites were lysed out of HFFs, urea-solubilized, and immunoprecipitated with bead-bound affinity-purified anti-TgSkp1 (pAb UOK75). The enriched preparation of TgSkp1 was eluted with triethylamine, reduced and alkylated, trypsinized, and analyzed by reverse phase-HPLC on an LTQ-XL Orbitrap MS. Extracted ion chromatograms showed coelution of a doubly charged (m/z 1436.6464) and a triply charged ion (m/z 958.0983) corresponding with a Δ mass of 0.56 ppm, to the predicted tryptic TgSkp1 peptide ¹⁴⁵IFNIVNDFT(HyP)EEEAQVR¹⁶¹ bearing a pentasaccharide with composition Hex3dHex1HexNAc1 (A). B, CID fragmentation of the doubly charged precursor ion yields a sequential loss of monosaccharide residues corresponding to Hex, Hex, dHex, Hex, and HexNAc, indicating the presence of a linear pentasaccharide. C, inspection of the full CID fragmentation spectrum shows b- (blue annotations) and y- (red annotations) ion series that match the predicted peptide sequence, as illustrated in the inset, and demonstrate that the glycan is linked via a hydroxylated derivative of Pro-154. Peptides with residual sugars are annotated in green.