I am presently researching the process of glycation in proteins and aiming to assess the effects of different sugars on proteins. My goal is to precisely identify the specific molecular alterations that occur when proteins interact with high levels of sugars. I would greatly appreciate any assistance in this endeavour. Thank you.
To get at that level of detail will require sophisticated techniques. You could start by doing intact protein mass spectrometry to measure the increase in mass caused by treating the protein with sugar. That will tell you how many sugar units were added. Next, you could do proteolytic digestion and peptide mass spectrometry to discover on which residues the glycosylation occurred. Ultimately, it will be necessary to determine the 3-D structure using one of 3 techniques: x-ray crystallography, multidimensional NMR (using isotopically modified protein), or cryo-electron microscopy (for very large molecules).
When I saw your title, I was going to suggest circular dichroism, but I guess that's too low resolution for you.
So what exactly are you trying to do? Is it interaction of proteins with soluble sugars or is it effect of change of glycosylation on 3D structure of your protein? In the first case you'll need NMR. In the other, any of the three methods listed by Adam should work in principle.
Tomáš Hluska, I'm trying to determine the changes in the 3D structure of the protein. I have tried Circular Dichroism, but those results only show that there has been a change in the structure; I wanted to know if I could determine where the change is happening in the protein.
A very quick-and-dirty technique is to compare the proteolytic peptides from proteins with and w/o modification, using western blots. Decades ago, I have done that with Na/K-ATPase where certain modifications switch between E1 and E2 conformation and thus increase sensitivity to trypsin and change the fragment pattern. At least, this should tell you whether there are significant changes in tertiary structure. And it is cheap to do, if you have an antibody (ideally, either against the N- or C-terminal end).
Of course, MS is much more sensitive, if that equipment is available.
I suggest using the most promising technique as HDX MS...glycosylation/glycation-dependent change at its high-order structure can be tracked by analyzing deuterium uptake which is elucidated by immobilized pepsin digestion and following the proteolytic peptide mapping...If it is not possible PMF analysis before and after deglycosylation can indicate the site-specific modification. If you have a full sequence then it would be easy to modified region at 3-D structure...I am not sure but pymol may be the solution at this stage...
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