A Unique Workflow for Glycoprotein Characterization from Sample Preparation to MS/MS Spectral Interpretation

摘要

Protein glycosylation is a complex dynamic post-translational modification, which is used by an organism to regulate a number of important functions. Variable composition, linkage, branching and anomericity of the constituent monosaccharides in combination with the general heterogeneity due to the indirect, non-template control of their biosynthesis are the basis of the structural complexity of glycoprotein glycans. This poster presents a complete workflow for glycoprotein characterization comprising sample preparation for glycan release, glycan separation using graphitized carbon separation coupled to MALDI spotting, automated MS and MS/MS analysis MALDI-TOF analysis and spectral interpretation with SimGlycan® Software. The workflow has been first optimized using commercially available immunoglobulin G from human serum and a glycan library of defined bisecting and none-bisecting glycan structures. The IgG sample contains both different protein sequence isoforms (IgG1-3) and different glycan isoforms. The results demonstrated that, the RGSPS kit with InstantAB™labeling provides a fast and robust glycan release and labeling method including sample clean up in less than 2 hours prior to mass spectrometric analysis. The combination of the LC MALDI Workflow with the nanoflow hypercarb column enables better glycan separation and therefore more specificity for isomeric glycans structures characterization. High energy CID MS/MS spectral comparison of the released and/or labeled glycans with the control samples provides a greater number of MS/MS fragments than electrospray CID MS/MS spectra or MALDI post source decay (PSD) spectra for more detailed information. Next, this robust workflow will be tested and expanded on therapeutically interesting antibodies. Studying the heterogeneity of glycosylation patterns obtained from cell culture influences can help understand efficacy, binding affinity, specificity and pharmacokinetic properties.