I have a problem about identifying a glycoprotein which I isolated from whole cell extract using standard immunoprecipitation method with special antibody against this unknown antigen. I perform trypsin in gel digestion and LC-MS to identify this protein. However, I try several times to search the matching results using my MS data and get nothing reliable. I am sure this protein is highly glycosylated and want to know whether I should remove the glycosylation modification before digesting the protein and how I cleave the N-link or O-link glycoside bond. Thanks very much and feel sorry for my English.

Hi,
What kind of detection system after running the gel are you using?
Sypro Ruby or Silver staining?

Thanks for reply. Actually I performed silver staining to visualize the unknown antigen after immunoprecipitation. Because it is a highly glycosylated protein, I can hardly see the clear band on the gel. So, I just cut the gel at the position corresponding to the Western Blotting results and get them digested. I also do not know whether I have got enough protein for LC-MS since the glycosylated protein can not be readily stained using common staining methods. I just believe I obtain enough quantity according to the western blotting results. What do you think is the problem?

Based off my experience you defiitely should remove the glycosylation. N-linked can be removed by treatment with PNGase F (commercially available). Removal of O-linked sugars is more difficult, but can be done by various glycosidases (you need to know the types of linkages of course) or potentially Beta-elimination chemistry (should be simple to find references). Glycopeptides do not fly in LC-MS and can inhibit trypsin digestion. Also do you know how many Arg and Lys groups are in your protein or is it a complete unknown?

Rb