In-depth site-specific N- and O-glycopeptide characterization
Kathrin Stavenhagen: Glycopeptide analysis remains challenging because of its sample heterogeneity resulting from the degree of glycosylation site occupancy (macroheterogeneity) and the different glycoforms attached to individual glycosylation sites (microheterogeneity).
With respect to the latter one, qualitative site-specific glycosylation information of glycoproteins can be obtained by unspecific protease treatment resulting in small amino acid stretches carrying the glycan. This improves determination of the glycosylation sites. However, detecting these glycopeptides by 1D-LC-ESI-MS/MS is challenging due to insufficient or irreversible retention on the stationary phase and thus multiple analyses with different LC-setups are required. Since biological sample amounts are usually limited, methods for acquiring comprehensive information in a single run are necessary.
To obtain qualitative information of the glycosylation site we set up an integrated C18-porous graphitized carbon (PGC)-LC-ESI-MS/MS platform, that retains hydrophobic glycopeptides on the C18 column and the more hydrophilic compounds of the flow-through on the PGC column. In combination with a high-resolution QTOF mass spectrometer, operated with combined high- and low-energy CID, this method represents an important step towards the comprehensive identification of multiple glycosylation sites and glycoforms from one injection.
(Glyco)peptides with different length and composition, resulting from unspecific protease treatment, were shown to feature different ionization efficiencies. Thus, for relative quantitation of the attached glycoforms and determination of the glycosylation site occupancy specific proteases, e.g. trypsin, resulting in uniform peptide stretches are used for glycopeptide analysis by C18-LC-ESI-QTOF-MS/MS.
This approach was evaluated using standard proteins and further successfully applied for the detailed site-specific characterization of biological samples such as IgG3 CH3 region and the heavily glycosylated human C1-inhibitor.In future our strategy will be advanced by application of high-resolution QTOF-ETD-MS/MS and implementation of new software tools.