Figure 2
From: Cross Reactive Material 197 glycoconjugate vaccines contain privileged conjugation sites
Conjugation site determination by tandem mass spectrometry.
(A) After Glu-C digestion a doubly charged precursor ion corresponding to glycopeptide 242KAKQYLEE249 carrying an ST3 epitope was detected and selected for fragmentation by CID and ETD. The red labeled K indicates the site of conjugation identified by tandem MS. (B) CID fragmentation resulted in a prominent Y-ion series indicating the loss of glucuronic acid and glucose, but no significant peptide backbone fragments. (C) ETD fragmentation of the peptide backbone confirmed peptide identity as well as K242 as the site of conjugation. (D) Extracted ion chromatogram of doubly charged ions of unconjugated and conjugated peptide 242–249 showing the separation of isobaric conjugate products by C18 LC [overlay of m/z = 504.40 (unconjugated peptide, black line), 568.85 (peptide with conjugated linker, blue line) and 928.45 (peptide with conjugated glycan, red line)]. Despite the proximity of the two potential conjugation sites, K242 was more effectively conjugated.
