A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex
A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex
复制标题
DOI：
10.1016/j.jasms.2007.04.013
复制
发表时间：
2007-08-01
影响因子：
3.2
通讯作者：
Brutschy, Bernhard
中科院分区：
文献类型：
--
作者：
Morgner, Nina;Kleinschroth, Thomas;Brutschy, Bernhard
激光诱导液珠离子解吸（LILBID）是一种新的基于激光的质谱分析方法，用于分析大的完整膜蛋白复合物及其亚基。在该方法中，离子从含有被洗涤剂溶解的疏水蛋白质复合物的水性微滴中被IR-激光解吸。该方法是高度敏感的，非常有效的样品处理，相对宽容的各种缓冲液，并检测在狭窄的，主要是低电荷态分布的离子。决定是否观察到完整复合物或其亚基的关键实验参数是激光强度：在对应于超软解吸的非常低的强度水平下，完整的复合物与少量洗涤剂分子一起被转移到真空中。在这些条件下，络合物的低聚状态（即，其四级结构）。在更高的激光强度下，复合物被热分解成亚基，任何残留的去污剂被剥离以产生多肽的真实质量。所研究的模型复合物来自土壤细菌Paracoccus acetificans的呼吸链，包括复合物III（细胞色素bc（1）复合物）和IV（细胞色素c氧化酶）。这些是充分表征的多亚基膜蛋白，单个疏水亚基由多达12个跨膜螺旋组成。
A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.