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

2. Introduction b b Glycosylation is the most common and versatile post-translational modification in high organisms, carbohydrates covalently bind to polypeptide backbones. b b Glycosylation performs 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” b b 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 b b Full characterization of PG-C: Glycosylation (N-linkage, O-linkage); Phosphorylation. b b 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 MW Carbohydrate detection Lectin blotting Phosphate-antibody screen Further analysis Phosphate detection

8. Preliminary data of PG-C

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

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

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

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

13. Future Work b b 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 b b Apply the characterization protocol to more complex recombinant glycoproteins b b 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;