post-translational modifications which increase molecular weight of the protein - (Feb/14/2008 )
I have about 20 spots on my 2-D gel which are all identified by MALDI-TOF as the same protein. I can understand that some times proteins undergo post-translational modifications which change their PI or a little bit their molecular weight but some of these spots are way far from their theoretical MW; the theoretical MW is 54 kD but there are some spots at about 70 kD. I haven't been able to find any post-translational modification which could explain these results. I have attached an image of my gel which shows some of those spots with numbers 1, 2, 3, 4, 5, 6.[attachment=4237:gel_picture.jpg]
acetylation or glycosylation?
I don't see the attachment. Also
some proteins such as Pol II which have many phospho sites
can run as a smear on a 2D gel. If a protein has say 20 phosphorylation sites
you can get many combinations of phosphorylation and therefore many spots.
BUT, if the protein you find on your gel in keratin you may have other problems.......
I'm guessing 6 is the apo protein, for want of a better identifier. 1-5 look like mixed glycosylation products, with maybe phosphorylation, making the proteins shift left in their pI. What that means for 7-9, I have no idea. Doesn't the MS give any hints as to the identity of the additional mass?
thank you all for the responses, actually never mind about 7, 8, 9 these are different proteins the story is about 1, 2, 3, 4, 5, 6. the mass-spec results tell that they are all the same protein all are ATP-synthase beta subunit. and I think the mass-spec results are quite solid and strong for these spots, not a big deal about 4,5 they are very close to the theoritical size of the protein 55 kD even not much concerned about 6 maybe protein degradation, but 1,2,3 and even the row of spots above them their is no post-translational modification which can satisfactorily explain it. "Swanny" and "the bearer" do you think glycosilation can change the protein size about 25-30 kD even in its most sever form? is there any way I can find it out?
somewhere here suggested phosphorylation (or sulphatation) may contribute to increase size but not to this extent alone; more likely are glycosylation and/or acetylation;
there are kits (f.i. NEB) of O- or N-deglycosylation kits; I am not for familiar for kits of deacetylation; you may find a suitable deacetylase; run this enzymes and check size again
Absolutely, glycosylation can radically change the MW of a protein, particularly N glycosylation. It can make highly branched glyco-trees that can make up to 80% of the total protein mass (this is, however, an extreme amount of sugar). As the Bearer said there are kits to test for the two glycosylation types. All you do is digest your protein with each enzyme separately, and run a gel of the reactions as well as undigested. If you can identify the protein with a Western, a 1D gel will be enough to show the loss of the glycosylation. The MW will drop to the expected 54kDa, so you can tell which type of glycosylation you have. If you want to dig deeper into the sugar, there are specialist glycobiology groups who will be able to help.
All the best,
Thanks Swanny, thanks the bearer, these information are very helpful.