Mass Spectrometric Analysis, Automated Identification and Complete Annotation of O-Linked Glycopeptides

Complex mixtures containing O-linked glycopeptides bearing SA1–0GalGalNAc structures, or single GalNAc units were subjected to collision-induced dissociation (CID) and electron transfer dissociation (ETD) analysis on a linear ion trap-Orbitrap mass spectrometer and the resulting data was analyzed using the Protein Prospector software. An overview of the structural information provided by the different fragmentation techniques, as well as their limitations, is presented. We illustrate the importance of the complementary information in the mass spectrometry survey scans as well as the different tandem mass spectrometry techniques. We also present some unique features offered by Protein Prospector that are advantageous in glycopeptide analysis: (i) considering a modification that will produce a neutral loss, without “labeling” the original modification site; (ii) merging CID and ETD search results; (iii) permitting the comparison of different modification site-assignments. Although these data were obtained from secreted glycopeptides, the observations and conclusions are also valid for the intracellular regulatory O-GlcNAc modification.