Figure 6.

Analysis of _SHCPS target antigen 1 and TetH_c-_SHCPS glycoconjugate. (A) MALDI-MS of _SHCPS target antigen 1. (B) ¹H NMR spectrum of _SHCPS target antigen 1. ¹H NMR spectra were acquired in D₂O with the HOD signal at 4.75 ppm serving as the internal reference. (C) ¹³C NMR spectrum of _SHCPS target antigen 1 with 1,4-dioxane (67.19 ppm) served as the internal standard. (D) MALDI-MS revealing 12 discrete peaks reflecting the incorporation of between 1 and 12 _SHCPS hexasaccharides per TetH_c protein as indicated above each peak. Unconjugated TetHc would be present as a peak at approximately m/z 53 000 as indicated. The increments match the expected mass of the hexasaccharide and linker following incorporation using the selected chemistry (1524 Da per hexasaccharide incorporated). (E) SDS-PAGE with subsequent Coomassie staining demonstrates the increased molecular weight of the TetH_c-SHCPS glycoconjugate compared to unconjugated TetH_c protein. No unconjugated TetHc was visible in the glycoconjugate. Loading was with 3.2 μg of TetH_c per lane and 3.4 μg and 6.8 μg of conjugate in the left and right lanes respectively.