able to identify the 211 form of the T45 peptide (211-T45) at a retention time of 15.57 minutes. As would be expected from such a complex mixture, a number of co-eluting ions were observed. However, the triply-charged 211-T45 ion at m/z 1064 was easily identified. The base peak ion chromatogram and full-scan mass spectrum for the targeted glycopeptide are shown in Figure 4.

The signal intensities for the doubly- and triplycharged glycopeptides (m/z 1595 and m/z 1064, respectively) were less intense than those for co-eluting peptides because glycosylated peptides typically have lower ionization efficiencies than unmodified peptides. The use of Dynamic Exclusion allowed for automated analysis of these low-intensity glycopeptides. The LCQ Deca XP was operated in Data Dependent Ion Tree mode, where the most abundant ion in the MS2 spectrum was selected for MS³, and the most abundant MS³ product ion was chosen for MS⁴. Figure 6. MS³ on the 210-G, GlcN ion. The dominant ion in the spectrum, the T45 peptide +GlcN at m/z 1333, was selected for MS⁴.

Figure 7. MS⁴ on the T45 +GlcN ion at m/z 1333.
MS² through MS⁴ spectra are presented in Figures 57. The MS² and MS³ spectra generated a heterogeneous collection of glycosylated product ions. GlcN was not easily cleaved from the peptide, so it serves as a marker to identify the site of glycosylation. The CID energy was sufficient at the MS⁴ stage to begin fragmenting the T45 peptide and to confirm the peptide sequence.<
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