Protein-Ligand MS

[Dakota Hamill]

I asked google this last night but didn't want to get into another trip down the journal article rabbit hole trying to chase more info when I figured it might be easier to just ask 3,500 people instead.

I was wondering if anyone knew if there was a technique that allowed the identification of a protein bound ligand without knowing the exact mass/structure of the ligand but while knowing the proteins sequence/mass/fragmentation pattern.

Basically, the protein would serve as a scavenging agent to selectively capture a particular compound which hopefully binds irreversibly, and can then act as a concentrated source of said ligand. 

If the ligand could be "washed out" of the protein without modification after the protein had been purified, fantastic, but if not, I was wondering if there was a way to see what ligand a protein's active site was bound to via LCMS or some other method, given in depth knowledge of the protein.  Perhaps you could subtract the fragmentation pattern of the protein since that is known and be left with the fragmentation pattern of the ligand, etc.  

I guess that's what predictive modelling of protein active sites with computers tries to show....a "guess" at the ligand that fits into the active site, but was wondering if there was perhaps a different way using a protein/ligand complex instead of guessing with software.

Ok thanks

[Nathan McCorkle]

x-ray crystallography?

Otherwise quickly I'm thinking:
get mass spec of pure unbound protein

mix protein with sample, have some way to purify protein from bulk,
get mass spec of pure protein+ligand... do analysis on computer to
subtract the original signal.

there are tags for doing protein purification... HIS tags, some other
stuff that isn't coming to mind... or even antibodies (but they'd also
need to be pure, so it would be sort-of double work, and not cheap)

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-Nathan

[Dakota Hamill]

Yeah actually I have no idea why I even wanted to keep the ligand in the protein, that's a headache, I wasn't thinking.

Purifying out the protein then trying to wash whatever ligand is still bound in it then just doing LCMS on that is much easier.  I don't care about the protein really, I just want something to pull a particular compound out of a cell without fractionating an entire cell lysate solution.  There's a pretty good chance the ligand wouldn't even stay bound in the active site anyway through all the steps in just trying to purify the protein.  Nevermind.