http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Life Science Discussion en-us sci7feed@sci7.nojunkorherepleasespam.com hourly 1 2005-05-05T12:00+00:00 http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html
Our argue is endless. How do you think, can acrylamide react with -COOH during alkylation process? Or alkylation specific for -SH group only?

Thank you a lot!]]> Fri, 27 Nov 2009 05:26:09 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html What do you think, can alkylation of protein by acrylamide affects PTMs (glycosilation, particularly)?
We could not explain phenomenon of decreasing of quantity of pI modification of protein of interest after alkylation.
And i did not such information.
Thank you for your suppositions!]]> Thu, 26 Nov 2009 10:45:39 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html ]]> Wed, 25 Nov 2009 09:29:40 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html
Thank you, Rajiv Dua! Very interesting article!

Brilliant thoughts!]]> Wed, 25 Nov 2009 02:35:59 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function.
Albert S B Edge
Harvard Medical School and Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA. Albert_Edge@meei.harvard.edu

With Regards,
Rajiv]]> Sat, 21 Nov 2009 01:02:40 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html
Thank you a lot for advise!! Very interesting articles!

Have a good day!
]]> Thu, 19 Nov 2009 13:02:07 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html This protocol is for removal of O-glycans only it will not remove N-glycans. Im not a MALDI expert by any means, so Im hesitant to advise you further. You're best bet is to grab some of Anne Dell or Julie Leary's recent papers and read through the methods. Those 2 are the world experts in analyzing glycan structure.
Rus
]]> Wed, 18 Nov 2009 09:00:59 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html
Could you advise me? Will i be able to use your protocol for MALDI analysis? And could i to determine glycoside structure on MALDI?

We are going to research O- and N-glycosiadtion of protein. But we have never done it. I looking for suitable protocol for geglycosiadtion and futher structure analysis protein and glycans.

Thank you!]]> Wed, 18 Nov 2009 07:03:41 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html I dont recommend in gel beta elimination. The high pH will make a mess. The polypeptide backbone will be unharmed if you use the conditions described in my basic protocol. The C18 reverse phase column will separate the sugars from the polypeptide allowing you to easily trypsin digest into peptides to analyze.

Rus]]> Tue, 30 Jun 2009 09:44:09 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Thank you for your reply. I am intending to try to do in gel beta elimaination. I am just wondering whether do you know whether the process will cause the lose of my protein after the release of the O-link as I don't really want to analyse the O-link, all I want is to look at the cleavage of my protein.
What is the purpose of the clean up with C18? I am afraid that the removal of O-link will cause the release of the protein backbone together with it.
Hope to hear from...]]> Mon, 29 Jun 2009 18:37:56 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html It seems to me doing the experiment the way you outlined may work, but consider this. It is likely that the glycosylation is interfering with the trypsin digest by simply preventing access to the peptide backbone through streric means. This would mean you get a lot less peptide coverage and diminish the chances of getting peptides that fly in MS and are readable, which is exactly your problem not enough sequence coverage correct?
So perhaps, you should beta-elimi...]]> Tue, 16 Jun 2009 11:04:56 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Thank heaps for the paper and the advice. I have another question. Here is what I am going to do;
1) I will run the SDS-PAGE gel with my recombinant protein and stain with coomassie blue
2) I will cut the bands, destain and proceed with DTT reduction and alkylate with iodoacetamide and follow by in gel tryptic digest. Then dry my peptide
3) Then, i will do the beta elimination and clean up with C18 spin columns before the LC/MS.
Do you think this is a good idea? or shoul...]]> Tue, 16 Jun 2009 01:57:26 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html
Remember that O-glycosidases cleave only unsubstituted Gal-ß(1-3)GalNAc-alpha disaccharides attached to the serine or threonine residues of glycoproteins. That means you will have to treat with a sialidase and/or Endo-O-glycosidase prior to using the O-glycosidase to remove all the terminal sugars from the O-glycans of the O-linked sugar. That is...]]> Thu, 11 Jun 2009 10:47:56 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html I have been wanting to reply to you but I have been busy lately. Thanks a lot for the protocol, however, I do not have a lot of protein to play around with, I have a recombinant protein of maximum of 4ug and dissolve in PBS. My protein is heavily O-linked glycosylated with only one N-linked. Therefore, I thought I might just use O-glycosidase to do it. So, I will be deglycosylating the protein and run the 1D SDS-gel before cutting the band and do tryptic digest on that before LC/...]]> Thu, 11 Jun 2009 00:51:55 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Did the protocol help? Havent heard anything back from you.

Rus]]> Wed, 10 Jun 2009 16:12:33 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Took me a bit to dig it up.

Basic protocol
BEMAD (β-elimination followed by Michael addition with DTT)
1. Resuspend peptides in 1% triethylamine, 0.1% NaOH, 20% EtOH, 10 mM DTT, pH 12. Incubate at 50OC for 2.5 hrs.
2. Quench with TFA (1% final conc)
3. Clean up with C18 spin columns. Elute with 0.1% TFA, 70% acetonitrile and dry

(Note this will add a DTT to the oxygen on site of glycosyation (Ser or Thr) it might also leave an OH grou...]]> Wed, 03 Jun 2009 12:36:40 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Thanks for the reply. If you don't mind, can u dig out the protocol for me. Thank heaps for your help.
Kit]]> Tue, 02 Jun 2009 08:45:52 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html If its O-linked then you can use beta-elimination to remove the sugars by breaking the bond to oxygen on the protein. Its pretty simple to do, if you need a protocol I can dig it up for you.

Rus]]> Mon, 01 Jun 2009 15:57:05 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html Sigma offers a deglycosylation kit for the proteins. check their life science catalogue for more information.]]> Sat, 30 May 2009 05:47:12 GMT http://www.scientistsolutions.com/t10720-deglycosylation+of+heavily+glycosylated+protein.html I have a problem with LC/MS on heavily glycosylated protein as the sequence coverage is relatively low. Therefore, i will be trying to deglycosylate the protein. What is the best method and where can i can the method from? Will the PGnase F affect the LC/MS result as it may be the enzyme that get sequence rather than my protein.
Kit]]> Thu, 28 May 2009 01:18:15 GMT