[Babcock_Hall]

We have some samples that came from protein crystals on which we collected data  The protein was chemically modified by the addition of one -CH₂PO₃ group putatively to a single cysteine reside; however, showing this by LC/MS of the trypsin-derived peptides has not been successful, despite repeated attempts.  Whole-protein mass spectrometry does show an increase in mass.  After diffraction data were collected, the crystals were dissolved and frozen.

We would like to show that the crystals still contain the modification.  It occurs to me that the retention time in RP-HPLC or the mass spectrum of the protein in these samples would be useful in showing that the protein that was crystallized was or was not identical to the protein prior to crystallization.  However, the protein is generally crystallized in the presence of a high concentration of high-MW polyethylene glycols.  Therefore, we need to remove the PEG from the protein and possibly concentrate.  I was thinking of ZipTips.  Does anyone have suggestions?

[Yggdrasil]

Ideally, your diffraction data should show the presence of the modification.  If you build a model of the structure without the modification then look at the 2Fo-Fc map, you should see some electron density off of the cysteine if the modification is actually present.

I've never tried to recover protein from crystals after data collection.  Remember that crystal will suffer from radiation damage, so data collection could definitely change your protein.

Alternatively, you could try an Ellman assay to quantify the number of free, unmodified cysteine residues in your protein before & after modification.

[Babcock_Hall]

I have wondered about the effects of radiation damage, but it seems to me that we have to try this or grow more crystals solely for the sake of dissolving and analyzing them.  We perform the Ellman assay routinely before and after the modification.  It shows a sharp drop, but not to zero.  The alkylating agent hydrolyzes quickly, making 100% modification difficult to achieve.  Then we purify by CEX-HPLC.  There is density beyond the sulfur residue, yet repeated attempts to solve the structure have not shown the modification.  Our collaborators have suggested that the group is disordered.  BTW, a different protein with the same modification was much better behaved in terms of MS.

[Yggdrasil]

What ionization method are you using for LC/MS?  Is it possible that the modification is lost during ionization?  LC/MS methods usually use harsher ionization methods than whole protein methods, which may explain the discrepancy you're seeing.

[Babcock_Hall]

We used ESI (electrospray ionization), and our collaborators used nanoES.  We are considering repeating the peptide work with a different protease, but I am not sure how to select one.  We have used phosphopeptide work as the basis for our reasoning in the past, but of course we have a phosphonate group, not a phosphoester.

[Babcock_Hall]

I like DeepView, but the visualization program is not the issue.  The problem is that there is little density for the phosphonomethyl group.  Either the group is not there, or it is highly disordered.