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The analysis of N- and O-glycopeptidesremains challenging dueto the microheterogeneity （different glycoforms attached to one glycosylationsite） and macroheterogeneity （site occupancy） of the glycoprotein.Trypsin is by far the most commonly used protease in glycoproteomicstudies; however, it often results in long peptides that can harbormore than one glycan which may hamper site identification. The useof unspecific proteases such as Pronase can largely overcome thisproblem by generating glycopeptides with a small peptide portion.While the resulting glycopeptides are very useful for tandem massspectrometric investigation, the analysis with conventional 1D-LC-ESI-MS/MSapproaches can lead to incomplete glycosylation coverage because ofthe very heterogeneous physicochemical properties of the glycopeptidesdepending on the peptide sequence as well as the size and chargesof the glycan moiety. Here, we describe a universal workflow for site-specificN- and O-glycopeptide analysis of Pronase treated glycoproteins withintegrated, sequential C18 reverse phase and porous graphitized carbon-LC-ESI-QTOF-MS/MSemploying a combination of lower- and enhanced-energy collision-induceddissociation. The approach was evaluated on glycoprotein standardsand also applied to investigate the glycosylation of human IgG3 providingdetails on the hitherto uncharacterized glycosylation site Asn392of the CH3 domain. This analytical tool can be appliedto a variety of glycoproteins for site-specific N- and O-glycopeptideanalysis, resulting in a good glycopeptide coverage within a singlesample run and, thus, requiring only small amounts of sample.
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