It sounds like upon binding with the ligand, your receptor is undergoing a conformational change which is exposing an underlying pocket of basic residues. My best guess is that the conformational change is due to alteration of the ionic or pH environment of the receptor. You may consider evaluating the sample in varying salt concentrations or physiologic pH values to see if you can bias towards a larger population of the subspecies or extend its length of time. Alternatively possibilities include something small such as a phosphorylation event (which may not be detected as a change in mass depending upon technique) or a cleavage event (although unlikely since you are likely to disrupt the interactions of cleaved products on a 2D gel). 

Hope this helps.

Chris

Thank you, Christopher. Phosphorylation would cause an acidic shift, but here we see a shift towards the very basic end (pH 10). We know that our protein gets phosphorylated in response to ligand binding due to initiation of downstream signalling, and we are also detecting acidic shifts that presumably reflect the phosphorylated species. Also, within the strongly denaturing environment provided by the 2D rehydration solution (urea + thiourea + detergent), it is unlikely that we would be able to observe differences in conformational changes on the 2D. So I am puzzled with regards the type of posttranslational modification that might have taken place and cause a basic shift. 

How about sumoylation?

Is this basic shift only happening on ligand binding? At what pH is the incubation? Or does the basic shift also happen with incubation with the buffers? Is the shift quantitative in 10 minutes?

Assuming it is buffer induced, some of the possibilities include N-terminal pyroglutamation; succinimide intermediate in the process of deamidation; esterification of carboxylic groups of Asp and Glu residues. 

You also mention the basic end at pH 10. What is the pI before this reaction is happening? What mass analysis technique you are depending on for apparently no shift in MW?

how about an allosteric regulator? post translational modifications are covalent modifications, allosteric modifiers typically alter protein structure through electrostatic interactions 

Agree with Christopher above that conformational change could still be a cause. In the un-affected species (ie no basic shift) how close is the pI you observe in 2D to the theoretically predicted pI (based on sequence/residues)? We recently found (and published) that for an enzyme we make, conditions that we assumed would be totally denaturing were in fact, not, and there was still structure there even in 8M urea. Worth checking to rule it out?

Dear all,

Thanks for all replies. I will provide some more information on the actual experiment:

1. I am working with CHO cells stably expressing the receptor I am interested in.

2. I either treat the cells with serum-free medium or the ligand, for 10 minutes.

3. The cells are lysed, and I IP the receptor of interest and process the samples for 2D western blotting. I should mention that prior to dilution of the samples with rehydration buffer containing 8M urea/2M thiourea, we elute in 4X LDS/2X reducing agent buffer at 98 degrees for 10 minutes, and given the set-up of the system, we are fairly certain that the shift is not purely a result of a conformational change and therefore incomplete denaturation.

4. It is a well-studied receptor, and we know that phosphorylation at multiple residues is a major posttranslational modifications in response to ligand binding. However, when we compare the receptor migration patterns across the two conditions - in addition to shifts corresponding to multiple phosphorylation events - we do observe a subspecies migrating around pH = 9 (we are using non-linear ZOOM IPG strips). The computational prediction of the pI is not very informative because the receptor is heavily glycosylated which we cannot account for in our in silico calculations. Receptors from cells treated with media migrate below pH 7 (there are several species), and ligand stimulation results in the appearance of several species that migrate around pH=4 (presumably due to phosphorylation which we know happens, and we have also blotted for it).

Hence why I was wondering about the basic shift. We will eventually follow these initial observations up with mass spec, but perhaps someone has an idea about a putative post-translational modification. Sumoylation is a good point if it results in a basic shift. Again, the vertical migration of the receptor is not altered, though.