Characterization of Recombinant Glycoprotein by Mass Spectrometry

1. Characterization of Recombinant Glycoprotein by Mass Spectrometry Min Xie Spring, 2001

2. Introduction • Glycosylationis the most common and versatile post-translational modification in high organisms, carbohydrates covalently bind to polypeptide backbones. • Glycosylationperforms critical biological functions in protein sorting, immune recognition, inflammation and other processes ----- Incomplete processing of carbohydrates causes serious diseases in humans, such as “carbohydrate deficient glycoprotein syndrome” and “congenic dyserythropoietic anemia” • Two types of carbohydrate chains are commonly present in glycoproteins: N-linked and O-linked

3. Typical N-linked Glycan Structures

4. Some O-linked Glycan Core Structures Core structures 3-5 have the same MW, which is different from the other two structures

5. Phosphorlyation

6. Goals • Full characterization of PG-C: Glycosylation (N-linkage, O-linkage); Phosphorylation. • Use the optimized analytical methods developed during characterization of PG-C to study other recombinant glycoproteins.

7. Flow Chart for Characterization of PG-C PG-C Database search MALDI-MS Lectin blotting Phosphate-antibody screen Further analysis MW Carbohydrate detection Phosphate detection

8. Preliminary data of PG-C

9. Analysis of N-linked Glycans PG-C Primary structure Lectin blotting Trypsin digestion GNA: terminally linked mannose; SNA: sialic acid terminally linked α(2,6)-galactose or GalNAc PNA:galactose-β(1,3)- GalNAc Affinity Chromatography N-glycopeptides Deglycosylation LC-MS/MS N-linkage sites

10. Analysis of N-linked Glycans (conc.) PG-C On-target Endo-H Deglycosylation PNGase-F Deglycosylation C-graph solid phase extraction Derivative method MALDI-MS MW of N-glycans CCSD database searching Sequential exoglycosidase digestion Primary structure

11. Analysis of O-linked Glycans PG-C N-Deglycosylation (PNGase-F) Lectin blotting Microcon microconcentrators or LC Trypsin digestion LC-MS/MS ( a splitter between LC and MS) MALDI-MS Linkage sites, primary structure

12. Analysis of phosphorylation PG-C trypsin digestion phosphate-antibody screen LC-MS/MS phosphate antibody column Compare detected peaks with database predicted ones phosphopeptides whether any peak shift at 80Da or 160Da MALDI-MS or LC-MS/MS phosphorylation sites

13. Future Work • Continue the characterization of PG-C. Where the O-linked glycosylation linkage sites are What the structure of O-linked glycans is Where phosphorylation linkage sites are • Apply the characterization protocol to more complex recombinant glycoproteins • Develop derivatization method: Whether it still works in “gel” environment

15. Lectin blotting

16. Derivatization Method A tag is formed at the reducing terminus of monosaccharides via reductive amination reaction. An aromatic amine attacks the carbonyl group in the open chain form, forming an schiff base, which is comparatively unstable and is reduced to the secondary amine. Advantages ----can be detected by UV absorption or fluorescence, thus can be monitored during LC separation. ----increase MS signal abundance and easier to be interpreted.

17. Database search result of PG-C MW of mature chain is 36227 Da;