[Babcock_Hall]

We partially inactivated VHR phosphatase with phosphonomethyltrifluoromethanesulfonate, based on enzyme assays.  Our intention is to convert the active-site cysteine into -CH₂-S-CH₂-PO₃.  We saw that the partially inactivated (and presumably chemically modified) protein eluted later in hydrophobic interaction chromatogrphy (HIC) than the protein peak when there was no inactivation.  When our collaborators attempted mass spec on the peptide containing the active-site cysteine, the mass of the unmodified peptide was dominant, but there was some evidence of the modified peptide being present.  Therefore, our working hypothesis is that some unmodified protein co-chromatographed with the modified protein and the unmodified protein (having less negative charge) flies better in the mass spec experiment.  Another possibility is that the modified peptide undergoes preferential degradation under the conditions of the mass spectral experiment.  A third possibility is that our HPLC peak assignment is somehow mistaken.  Any thoughts on what the problem might be and how to proceed would be appreciated.

[Yggdrasil]

I don't know much about the phosphonomethyltrifluoromethanesulfonate reagent, but why use that instead of a more standard reagent to selectively modify cysteines (e.g. N-ethyl maleimide or 2-iodoacetamide)?  If you think charge is an issue, perhaps using one of these reagents instead may give better results.

Is the active-site cysteine the only cysteine in your protein?  It's possible that your reagent is modifying antoher cysteine to inactivate the protein.

Finally, how are you doing your mass spec?  ESI?  MALDI?  A labmate of mine was having trouble identifying the site where his protein was modified by a crosslinking reagent using ESI, but he got better results MALDI (though I'm not sure why that is, perhaps it's worth talking to an expert on protein mass spec for that info).

[Babcock_Hall]

Our goal was to create a stable analog of the cysteinyl phosphate intermediate, -CH₂-S-PO₃.  We cannot use maleimide for that reason, and iodoacetate has already been used to inactivate this enzyme (but not with the thought of making an analog).  Initially we used the native form of the enzyme, which has three other cysteine resides.  However, one of them might also become modified under some circumstances; therefore, we switched to a triple Cys-to-Ser mutant.  Our collaborator is using MALDI, but I need to increase my knowledge of protein and peptide mass spectrometry, so that I can better understand the advantages and disadvantages of each.

[Yggdrasil]

What's the selectivity of your reagent for cysteines?  It seems like it would also react with many other nucleophilic groups in the protein, so the inactivation observe in your enzyme assays may not be due to modification of the active site cysteine.  In the mass spec, do you see evidence of other peptides from your protein being modified?

[Babcock_Hall]

This is the third protein we have reacted with phosphonomethyltrifluoromethansulfonate (PMT), although the two previous ones were the same protein from two different species.  I have not seen much evidence for reactivity at positions other than cysteine.  We inactivate the phosphatase near neutral pH; therefore, lysines should mostly be protonated.  We did a protection experiment with a related reagent, phosphonomethylpentafluorobenzenesulfonate, but protection was incomplete, and we think a second cysteine may have been modified.  This reagent reacts in two ways, unfortunately, making it unsuitable for our purposes.  We probably need to put a protection experiment on our to-do list for PMT.