Mass spectrometric analysis of mercury incorporation into proteins for X-ray diffraction phase determination.
Heavy-atom incorporation is an essential and often rate-limiting step in the determination of phases for X-ray diffraction studies of protein structures. Until the present, there has been no practical method (short of the X-ray diffraction experiment itself) to judge the success and extent of incorporation. Here we show that mass spectrometry is an effective tool for determining the extent of heavy-atom incorporation in proteins. In particular, we demonstrate the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) for assaying mercury derivatization of cysteinyl thiol groups in proteins. Each of these mass spectrometric methods has advantages and drawbacks. ESI-MS provides a more accurate quantitative measurement of the extent of mercury incorporation, while MALDI-MS provides a useful lower limit to the level of mercury incorporation. Conversely, MALDI-MS does not require removal of excess derivatization reagents, salts and buffers, thus permitting facile analysis of single protein crystals as well as rapid, semiquantitative evaluation of the extent of protein mercuration. The approaches described in the present paper have contributed to the successful X-ray analyses of several noteworthy protein structures.
Cohen
S L
SL
Rockefeller University, New York, New York 10021, USA. steven_cohen@merck.com
Padovan
J C
JC
Chait
B T
BT
D003460:Crystallization; D003545:Cysteine / Q000737:chemistry; D008628:Mercury / Q000737:chemistry*; D008970:Molecular Weight; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014961:X-Ray Diffraction / Q000379:methods*
D011506:Proteins; D008628:Mercury; D003545:Cysteine