In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
近年来，质谱技术在膜蛋白结构与功能研究中被广泛应用。由于膜蛋白的跨膜结构域含有大量疏水性氨基酸，常常导致液质串联质谱检测的序列覆盖率较低，从而限制了质谱技术在膜蛋白结构与功能研究中的应用。文中利用人的整合膜蛋白维生素K 环氧化物还原酶为模型，优化胶内消化条件，建立了一种稳定提高膜蛋白质谱序列覆盖率的糜蛋白酶胶内消化方法。通过探索钙离子浓度、pH 值和缓冲体系对序列覆盖率、检测特异肽段的总数和类型以及特异肽段大小的影响，发现在5–10 mmol/L 钙离子浓度、pH 8.0–8.5 的Tris-HCl 缓冲液中，可以兼顾序列覆盖率和肽段的多样性。该方法可以使膜蛋白的质谱覆盖率达到80%以上，将在膜蛋白结构与功能、膜蛋白相互作用位点的鉴定以及膜蛋白与小分子药物结合位点的鉴定等研究中具有广泛的应用价值。.
Keywords: LC-MS/MS; chymotrypsin; in-gel digestion; membrane protein; membrane protein structure; sequence coverage; vitamin K epoxide reductase.