Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis
Supercritical fluid chromatography reduction of hydrogen/deuterium back exchange in solution-phase hydrogen/deuterium exchange with mass spectrometric analysis
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DOI：
10.1021/ac060693n
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发表时间：
2006-10-01
影响因子：
7.4
通讯作者：
Greig, Michael J.
中科院分区：
文献类型：
--
作者：
Emmett, Mark R.;Kazazic, Sasa;Greig, Michael J.
作为蛋白质（或蛋白质复合物）表面暴露的质谱探针，溶液相H/D交换的最大问题是在初始H/D交换被淬灭后H与D的反向交换。反向交换导致相关数据的丢失，也极大地阻碍了数据分析。以前，使用非常快速、冷（0-4℃）的HPLC来帮助减少反交换，但计算出的反交换平均仍接近30%。在本报告中，超临界流体色谱法取代HPLC法作为质量分析之前的脱盐/分离技术，与快速、冷的HPLC法相比，显著减少了反交换。
The single biggest problem with solution-phase H/D exchange as a mass spectrometric probe of surface exposure in a protein (or protein complex) is back-exchange of H for D after the initial H/D exchange has been quenched. Back-exchange results in loss of pertinent data and also greatly hampers data analysis. Previously, very fast, cold (0-4 degrees C) HPLC was performed to help reduce back-exchange, but calculated back-exchange still averages similar to 30%. In this report, supercritical fluid chromatography replaces HPLC as the desalting/separation technique prior to mass analysis, providing a dramatic reduction in back-exchange compared to the fast, cold HPLC methods.