Smith D L
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA.
Biochemistry (Mosc). 1998 Mar;63(3):285-93.
Amide hydrogen exchange rates, determined by NMR spectroscopy, have become an important tool that is often used to investigate structure and dynamics of small proteins. Recent developments in mass spectrometry and sample handling methods make possible measurement of deuterium levels at peptide amide linkages in polypeptides. The ability to make these measurements has led to development of the protein fragmentation/mass spectrometry approach for determining amide hydrogen exchange rates in short segments of intact proteins following their incubation in D2O. Partially deuterated proteins are proteolytically fragmented into peptides whose molecular weights are determined by on-line liquid chromatography/mass spectrometry. Deuterium levels, which are determined from the molecular weights of the peptic fragments, can be used to determine amide hydrogen exchange rates. Details of the protein fragmentation/mass spectrometry approach, along with a brief review of the theory of amide hydrogen exchange, are described. The ability to detect and locate minor structural differences in proteins by the protein fragmentation/mass spectrometry approach is illustrated using oxidized and reduced cytochrome c. These results show that oxidation of iron has little effect on the N- and C-terminal regions, but significantly destabilizes the interior regions of cytochrome c. The ability to detect localized unfolding in large proteins is illustrated with aldolase that was equilibrated in acid. Despite the success achieved by NMR spectroscopy for determining amide hydrogen exchange rates, mass spectrometry is advantageous because it permits studies of large proteins, requires only picomoles of protein, and provides a direct measure of structural heterogeneity.
通过核磁共振光谱法测定的酰胺氢交换速率，已成为一种重要工具，常用于研究小蛋白质的结构和动力学。质谱分析和样品处理方法的最新进展使得测量多肽中肽酰胺键处的氘含量成为可能。进行这些测量的能力推动了蛋白质片段化/质谱分析方法的发展，用于在完整蛋白质在D2O中孵育后，测定其短片段中的酰胺氢交换速率。部分氘代的蛋白质经蛋白酶水解成肽段，其分子量通过在线液相色谱/质谱法测定。由消化片段的分子量确定的氘含量，可用于测定酰胺氢交换速率。本文描述了蛋白质片段化/质谱分析方法的细节，以及对酰胺氢交换理论的简要回顾。使用氧化型和还原型细胞色素c说明了通过蛋白质片段化/质谱分析方法检测和定位蛋白质中微小结构差异的能力。这些结果表明，铁的氧化对N端和C端区域影响很小，但会显著破坏细胞色素c内部区域的稳定性。用在酸性条件下平衡的醛缩酶说明了检测大蛋白质中局部解折叠的能力。尽管核磁共振光谱法在测定酰胺氢交换速率方面取得了成功，但质谱分析具有优势，因为它允许研究大蛋白质，仅需皮摩尔量的蛋白质，并能直接测量结构异质性。
