characterization of recombinant glycoprotein by mass spectrometry n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Characterization of Recombinant Glycoprotein by Mass Spectrometry PowerPoint Presentation
Download Presentation
Characterization of Recombinant Glycoprotein by Mass Spectrometry

Loading in 2 Seconds...

play fullscreen
1 / 17

Characterization of Recombinant Glycoprotein by Mass Spectrometry - PowerPoint PPT Presentation


  • 88 Views
  • Uploaded on

Characterization of Recombinant Glycoprotein by Mass Spectrometry. Min Xie Spring, 2001. Introduction. Glycosylation is the most common and versatile post-translational modification in high organisms, carbohydrates covalently bind to polypeptide backbones.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Characterization of Recombinant Glycoprotein by Mass Spectrometry' - fabiano-knight


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
introduction
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

some o linked glycan core structures
Some O-linked Glycan Core Structures

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

goals
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.
flow chart for characterization of pg c
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

analysis of n linked glycans
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

analysis of n linked glycans conc
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

analysis of o linked glycans
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

analysis of phosphorylation
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

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

derivatization method
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.

database search result of pg c
Database search result of PG-C

MW of mature

chain is 36227 Da;