MASS SPECTROMETRY OF HEAVY ATOM INCORPORATION INTO PROTEINS

转换从蛋白质晶体获得的X射线衍射数据 进入三维结构需要确定 X光阶段 传统上，相位确定需要 在蛋白质晶体中包含重原子。 质谱 提供了一种分析重原子结合的实用方法 转化成蛋白质。 没有其他方法可用。 我们调查 使用MALDI和ESI-MS将汞掺入蛋白质。 之所以选择汞化试剂，是因为它们广泛用作 蛋白质晶体学中的重原子试剂 我们发现戏剧性的 汞化蛋白质的MALDI和ESI之间的差异。 电喷雾电离产生了最准确的，定量的 结果，虽然样品需要广泛的清理。 MALDI-MS 产生了更快的信息比ESI-MS因为样品净化 要求是最低的。 然而，尽管ESI-MS显示接近100%， 给定汞衍生的蛋白质的汞衍生， MADLI-MS一贯并且经常戏剧性地低估了 衍生化。 异常MALDI效应依赖于 MALDI基质、基质溶液的pH值和对汞的种类 试剂. 我们将结果之间的大部分区别归因于 能量“较冷”的电喷雾固有的差异 过程和“更热”的MALDI过程。 描述这项工作的论文 正在准备中。

Transforming X-ray diffraction data obtained from protein crystals into three-dimensional structure requires the determination of the X-ray phases. Traditionally, phase determination necessitates the inclusion of heavy-atoms in the protein crystals. Mass spectrometry provides a practical means of analyzing incorporation of heavy atoms into proteins. No other methods have been available. We investigated of the incorporation of mercury into proteins using MALDI- and ESI-MS. Mercurating reagents were chosen because of their widespread use as heavy-atom reagents in protein crystallography. We found dramatic differences between the MALDI and ESI of mercurated protein. Electrospray ionization yielded the most accurate, quantitative results, although samples required extensive clean up. MALDI-MS yielded more rapid information than ESI-MS since sample clean-up requirements were minimal. However, while ESI-MS showed nearly 100% mercury derivatization of a given mer cury-deriv atized protein, MADLI-MS consistently and often dramatically underestimated the extent of derivatization. The anomalous MALDI effects were dependent on the MALDI matrix, the matrix solution pH and on the type of mercurating reagent. We attribute much of the distinctions between the results to the differences inherent to the energetically "cooler" electrospray process and the "hotter" MALDI process. A paper describing this work is in preparation.