Figure 5
From: Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites
Comparison of conjugation efficiency on peptides 231–314 and 460–535 shown for four samples carrying different glycoepitopes.
In the SurveyViewer (retention time vs. m/z) of a (C4) LC-MALDI-MS experiment (left) intensities of the detected signals are plotted vs. their retention times and signal intensities in a gel-like representation, providing a quick overview on the number of conjugates present on each CNBr peptide (right). Spectra obtained for peptide 460–535 at a given retention time. Samples conjugated with the DSA-linker appeared in clusters with varying amounts of linker and glycoconjugates. Linker addition induced a shift of + 128 Da and minor shifts in retention time. A single glycoconjugate addition increased the mass by 848 Da (ST3), 333 Da (GLC), or 991 Da (PS1), respectively. Major differences in loading of free linker and glycans were easily detectable by this approach. ST3 batch-to-batch comparison revealed batch A containing up to one glycan conjugated to peptide 231–314 and up to two conjugated to peptide 460–535, whereas the loading was increased in batch B with up to two glycoconjugates attached to peptide 231–314 and up to three to peptide 460–535, respectively. The lower glycan loading in batch A correlated with stronger signals for linker conjugation. GLC was conjugated via a PNP-linker and showed no detectable free linker additions and therefore no clusters as seen for the DSA-conjugated peptides.
