Characterization of Recombinant Glycoprotein by Mass Spectrometry
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Characterization of Characterization of Recombinant Glycoprotein Recombinant Glycoprotein
by Mass Spectrometryby Mass Spectrometry
Min XieMin Xie
Spring, 2001Spring, 2001
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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
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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
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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
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PhosphorlyationPhosphorlyation
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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Database search result of PG-Database search result of PG-CC
MW of mature
chain is 36227 Da;