deglycosylation of heavily glycosylated protein

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kit
kit's picture
deglycosylation of heavily glycosylated protein

Hi all,

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

g a
g a's picture
Hello Kit

Hello Kit
Sigma offers a deglycosylation kit for the proteins. check their life science catalogue for more information.

R Bishop
R Bishop's picture
How is the protein

How is the protein glycosylated? N or O-linked?  If N then PNGase is your easiest option.  Yes you will detect it in the spectra.  However it shouldn't mask any unique spectra especially with LC-MS, since you can fliter out the spectra when you database search them.
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

kit
kit's picture
Hi R Bishop,

Hi R Bishop,

Thanks for the reply. If you don't mind, can u dig out the protocol for me. Thank heaps for your help.

Kit

R Bishop
R Bishop's picture
Hi kit,

Hi kit,
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 group if the DTT doesnt go on.  You will need to account for the mass difference in your spectra of course.)
 
As an alternative you can do this much milder treatment, but it is messy.
 
Resuspend you sample in a final concentration of 0.5M NaOH, 1M NaBH4  incubate overnight at 4C.  (I usually make up a 10X solution of the Beta-elimination solution and add it directly to my sample in the appropriate volume.  It will be slightly turbid.)
the next day quench the reaction with drops of 10N Acetic Acid and add a few uls of MeOH to prevent bubbles blowing out of the tube with your sample.  Check the pH with strips until it reaches pH 7.0 range.
 
Any questions?  Im here
Rus
 

R Bishop
R Bishop's picture
Hey kit,

Hey kit,
Did the protocol help?  Havent heard anything back from you.
 
Rus

kit
kit's picture
Hi Rus,

Hi Rus,
  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/MS. Do you think it is a good idea?

R Bishop
R Bishop's picture
Hmmmm.  I want to be careful

Hmmmm.  I want to be careful here and not over complicate what you are doing.  Here's a few things to keep in mind.
 
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 why you see deglycosylation "kits" that come with a series of enzymes such as the one that Argerine describes in this thread.
 
That being said, you might treat a tiny amount of your recombinant protein with the O-glycosidase and run it on a gel to see if you removed it, 100ng should show up on a Silver stain.  My hypothesis is that it wont do its job.  That is why the BEMAD method was developed. Being straight chemical attack on the O-glycosidic bond at peptide chain.  I can say that in my hands the BEMAD method is quite simple and works well.  You might give a look at the original papers by Lance Wells when he was with Gerry Hart, since they basically perform LC/MS/MS post treatment like you are attempting.
 
link to paper http://www.mcponline.org/cgi/content/abstract/1/10/791
 
Like I said, I dont want to influence you just advise on the possible outcomes.  These things get tricky with tiny amounts of a precious sample. 
 
Good luck
 
Rus
 
 
 

kit
kit's picture
Hi Rus,

Hi Rus,
  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 should i do the beta elimination before the tryptic digest, therefore it will be in gel beta elmination.

R Bishop
R Bishop's picture
Kit,

Kit,

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-eliminate after the ConA run.  Then gel purify the shifted band, then trypsin-digest, C18 clean up and LC/MS.  Again dont let me influence you too much.  Im just concerned you are losing a ton of peptides the way you outlined the experiment.
 
Rus

kit
kit's picture
Hi Rus,

Hi Rus,
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 you soon.
Cheers,
Kit

R Bishop
R Bishop's picture
Kit,

Kit,
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

Tegrion
Tegrion's picture
Hi, Rus!

Hi, Rus!

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!

R Bishop
R Bishop's picture
Tegrion,

Tegrion,

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

Tegrion
Tegrion's picture
Hi, Rus!!

Hi, Rus!!

Thank you a lot for advise!! Very interesting articles!

Have a good day!

Rajiv Dua
Rajiv Dua's picture
Hi...i hope this article will

Hi...i hope this article will be useful to You..

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

Tegrion
Tegrion's picture
Hi!!

Hi!!

Thank you, Rajiv Dua! Very interesting article!

Brilliant thoughts!

R Bishop
R Bishop's picture
Yeah nice.  I havent seen

Yeah nice.  I havent seen that one.  Thanks!

Tegrion
Tegrion's picture
Dear colleagues!!

Dear colleagues!!

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!

Tegrion
Tegrion's picture
Hi!!

Hi!!

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!