Characterization of Characterization of Recombinant Glycoprotein Recombinant Glycoprotein

by Mass Spectrometryby Mass Spectrometry

Min XieMin Xie

Spring, 2001Spring, 2001

IntroductionIntroduction

Glycosylation is the most common and versatile post-translational modification in high organisms, carbohydrates covalently bind to polypeptide backbones.

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”

Two types of carbohydrate chains are commonly present in glycoproteins:

N-linked and O-linked

Typical N-linked Glycan Typical N-linked Glycan Structures Structures

NeuAc NeuAca 2,3 or 6 a 2,3 or 6

Gal Gal Gal Man Man Man 1,4 1,4 1,4

GalNAc GalNAc GalNAc Man Man Man Man Man

ß 1,2 ß 1,2 ß 1,2 a 1,3 a 1,6 a 1,2 a 1,3 a 1,6

Man Man Man Man Man Man

a 1,3 a 1,6 a 1,3 a 1,6 a 1,3 a 1,6

GlcNAc Man Man GlcNAc Man ß 1,4 ß 1,4

ß 1,4 ß 1,4 ß 1,4

GlcNAc GlcNAc GlcNAc ß 1,4 ß 1,4 ß 1,4 a 1,6

Fuc GlcNAc GlcNAc GlcNAc

N N N

Asn-X-Ser/Thr Asn-X-Ser/Thr Asn-X-Ser/Thr

Complex Hybrid High-Mannose

Some O-linked Glycan Core Some O-linked Glycan Core

Structures Structures

ß 1,3 Gal

ß 1,6 GlcNAc

Core 1: Ser/Thr – O – a GalNAc – ß 1,3 – Gal

Core 2: Ser/Thr–– O ––a – GalNAc

Core 3: Ser/Thr –– O ––a – GalNAc – ß 1,3 – GlcNAc

Core 4: Ser/Thr –– O ––a – GalNAc – ß 1,6 – GlcNAc

Core 5: Ser/Thr –– O ––a GalNAc –a 1,3 – GalNAc

Core structures 3-5 have the same MW, which is different from the other two structures

PhosphorlyationPhosphorlyation

GoalsGoals

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.

Flow Chart for Characterization Flow Chart for Characterization of PG-Cof PG-C

PG-C

Database search

MALDI-MS

MWCarbohydrate

detection

Lectin blottingPhosphate-antibody

screenFurther analysis

Phosphate detection

Preliminary data of PG-CPreliminary data of PG-C

500

550

600

650

700

750

800

35000 36000 37000 38000 39000 40000 41000 42000 43000 44000 45000

Series1experimental MW of PG-C @ 39760 Da

after enho-H treatment: 38560 Da

database predicted MW @ 36227 Da

m/e

Analysis of N-linked GlycansAnalysis 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 GalNAcPNA:galactose-β(1,3)- GalNAc

Analysis of N-linked Glycans Analysis of N-linked Glycans (conc.)(conc.)

PG-C

On-target Endo-HDeglycosylation

MALDI-MS MW of N-glycans

PNGase-F Deglycosylation

C-graph solid phase extraction

Derivative method

CCSD database searching

Sequential exoglycosidase digestion

Primary structure

Analysis of O-linked GlycansAnalysis 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

Analysis of phosphorylationAnalysis 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

Future Work 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

I

Immobilization ofprotein on the wellsof an immunoplate

II

Application oflectins, which bindto specific glycan

structure

III

Application ofsecond antibody,which binds to

lectins

IV

Development ofcolor reaction. (Theabsorbances of thewells are propor-

tional to the amountof lectin bound)

Lectin blottingLectin blotting

Derivatization MethodDerivatization 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 secondaryamine.

Advantages----can be detected by UV absorption or fluorescence, thus can be monitored during LC separation. ----increase MS signal abundanceand easier to be interpreted.

Database search result of PG-Database search result of PG-CC

MW of mature

chain is 36227 Da;