Fig. 2. Characterization of rhamnosylated glycopeptides by nanoUHPLC-MS/MS. (A) Extracted-ion chromatographic elution profiles of synthetic glycopeptides 14 and 15, an equimolar mixture of 14 and 15, and Lys-C digested EF-P purified from P. aeruginosa or recombinant EF-P (from E. coli) with in vitro rhamnosylation by EarP. The extracted ion mass window was 349.86–349.87 m/z corresponding to the triply charged glycopeptide. (B) Ion-trap CID MS/MS analysis of the α-linked synthetic glycopeptide. (C) Ion-trap CID MS/MS analysis of the β-linked synthetic glycopeptide. (D) Ion-trap CID MS/MS analysis of the glycopeptide identified from P. aeruginosa purified EF-P. (E) Distribution of the diagnostic fragment ion [M + 2H-rhamnose]²⁺ normalized to the precursor ion intensity. **P < 0.01 (students t-test). All b- and y-type ions are annotated within 0.6 Da and represent the fragment containing the rhamnose glycan still attached via glycosidic linkage to the fragment ion.