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 th e determination of phases for X-ray diffraction studies of protein structur es. Until the present, there has been no practical method (short of the X-r ay diffraction experiment itself) to judge the success and extent of incorp oration. Here we show that mass spectrometry is an effective tool for deter mining the extent of heavy-atom incorporation in proteins. In particular, w e demonstrate the utility of matrix-assisted laser desorption/ionization ma ss spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (E SI-MS) for assaying mercury derivatization of cysteinyl thiol groups in pro teins. Each of these mass spectrometric methods has advantages and drawback s. ESI-MS provides a more accurate quantitative measurement of the extent o f mercury incorporation, while MALDI-MS provides a useful lower limit to th e level of mercury incorporation. Conversely, MALDI-MS does not require rem oval of excess derivatization reagents, salts and buffers, thus permitting facile analysis of single protein crystals as well as rapid, semiquantitati ve evaluation of the extent of protein mercuration. The approaches describe d in the present paper have contributed to the successful X-ray analyses of several noteworthy protein structures.