Chromatography Forum

Dear Fellow Chromatographers,

I'm trying to develop a method to enrich glycoproteins from non-glycosylated proteins. I'm currently using a HILIC column with ammonium acetate and acetonitrile. The gradient starts with high ACN and then moves to high buffer compositions.

At 70 to 80% ACN starting compositions, the proteins appear to ppt out in the sample! With 50% ACN they are appear ok, but the glycoproteins are retained poorly!

My question: Has anybody used HILIC for separation of proteins? (I know there is literature for peptides).

Has anybody used lectin affinity chromatography columns for glycoproteins? (I have not come across any literature so far!)

Why don't you use an hydrazine resin to enrich the glycoproteins?

Hydrazine resin? Could you please let me know whether there is any literature about this. Please let me know if this is available commercially.

Ruidi Aebersold introduced the method some years ago. Below I include an article from him but there are more from his and other groups. The hydrazine resin is available from several vendors (i.e. Bio-rad, Pierce etc) but there is no kit available (i.e. you will have to make your own reagents)... actually have a look around, as I haven't something might have been introduced lately.


Title: Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry
Author(s): Zhang H, Li XJ, Martin DB, Aebersold R
Source: NATURE BIOTECHNOLOGY 21 (6): 660-666 JUN 2003

A high percentage of acetonitrile is commonly used for protein precipitation, so I do not think that your HILIC method will have a lot of success. Maybe with another organic solvent like methanol you may have more success.

Kostas: how about boronate resins? Has this ever been used for glycoproteins? Could be even more specific....

mprajesh,

We just introduced new mixed mode HILIC/ion-exchange columns at Pittcon. You can increase amount of water up to 20% compare to traditional HILIC (70% vs. 90%) and this will help you with solubility issue You compounds will retain based on HILIC and cation or anion exchange mechanisms. The phase has both ionizable cation exchange and anion exchange groups. Multiple interactions can be adjusted by pH and buffer concentration:

http://www.sielc.com/Products_Obelisc.html


If you send us sample we can try it in the lab (free of charge)

Regards,

Vlad