用于膜蛋白质组分析的液相色谱-质谱联用新方法研究

Membrane proteins play an important role in a variety of cellular functions, ranging from signal transduction, subcellular compartmentalization, membrane trafficking, and protein secretion, in addition to their function in providing and maintenance of the structural integrity of membranes. Not surprisingly, the membrane associated proteins account for nearly 60% of pharmaceutical drug targets. Although we do not know the exact number of membrane proteins, it was estimated, following the sequencing of the human genome, that 20 percent of the human genome was membrane proteins. Effecient analysis of membrane protein and correct linking the structure and function of membrane protein will be hlepful to improve the successful rate of drug design. Unfortunately, membrane proteins are often underrepresented in proteomic experiments due to their low abundance and their hydrophobicity. Clearly, we need technologies for the study of the membrane proteome. Therefore, membrane protein analysis represents a major technological challenge in proteomics and analytical chemistry.　We recently developed a set of technologies, based on the proteomic reactor with pH fractionation, facilitating low-abundant protein identification and in particular membrane proteins.　The conventional reversed phase liquid chromatography is a dominant approach from peptide separation in LC-MS. However, the peptides from hydrophobic membrane protein might be tightly bound to the RP stationary phase by the hydrophobic interaction, and the extremely hydrophobic peptides cannot be efficiently elute off the RP stationary phase even using high percent organic solvent gradient. As the name implies, the major separation mechanism of SCX is electrostatic in nature, and high percent organic solvent in the pH buffer used in SCX separation can improve peptide solubility and reduce the hydrophobic interaction between peptides and SCX stationary phase. In this study, the on-line pH gradient eluted SCX-MS method will be develop for membrane protein identification, quantification and evaluation. This new approach will be a promising tool for drug membrane protein target discovery and for the potential membrane protein target/biomarker discovery to clinic diseases.

在人类基因组约3~4万个基因中，约有8千个基因是膜蛋白的编码基因，占20%以上。这些膜蛋白承担着至关重要的生物功能，并且目前市场上约60％的药物作用靶点为膜蛋白。对膜蛋白进行有效的分析，并对其结构与功能数据的正确把握，能够在很大程度上提高药物设计的成功率。然而，膜蛋白由于其丰度低以及疏水性强等物理化学性质，也成为当前蛋白质组学研究中的难点。在前期工作中，我们已经成功建立了膜蛋白质分级等一系列的样品制备方法，通过亚细胞分级等富集方法来解决膜蛋白丰度低的难题。在后续实验中，我们拟建立新的液-质联用方法（即在较高有机溶剂中，采用连续pH梯度洗脱的强阳离子交换液相色谱与纳升级电喷雾质谱直接联用）来提高膜蛋白质的检出率，希望能较好解决膜蛋白疏水性强的难题。通过本课题的研究，我们拟建立用于新药研发的膜蛋白质组鉴定、定量和验证方法，为药物膜蛋白靶点鉴定和疾病相关潜在药物膜蛋白靶点发现提供高效的研究手段。

在本面上项目的支持下，我们建立了新的在线pH梯度洗脱的强阳离子交换液相色谱-质谱联用方法（SCXLC-MS/MS），将其应用于蛋白质组学分析，并用标准蛋白、HuH7细胞总蛋白、N2a细胞总蛋白、N2a细胞膜蛋白样品对该方法进行了测试评估。该方法建立在纳升级液相色谱串联高分辨质谱系统上，使用纳升级毛细管强阳离子交换色谱柱以及纳升级电喷雾离子源。蛋白质酶解所得肽段在强阳离子交换色谱柱上通过pH梯度进行分离、洗脱，再经过电喷雾离子源离子化后，直接导入质谱进行分析。采用90 min的液相色谱-质谱检测程序，对来自总蛋白或膜蛋白的500 ng肽段进行分析，每一针都能够鉴定到5000多个肽段序列。将该pH梯度洗脱的在线强阳离子交换液相色谱-质谱方法与反相液相色谱-质谱方法进行对比，发现二者鉴定到的肽段序列重叠率低于30%，这表明两种方法具有互补性。该强阳离子交换液相色谱中肽段保留时间与其等电点具有较显著相关性，相关性系数> 0.4，这表明在该强阳离子交换液相色谱方法中肽段的洗脱与其所带电荷数相关。通过进一步对比分析，我们发现该强阳离子交换液相色谱-质谱方法与反相液相色谱-质谱方法相比，能够鉴定到更高比例的碱性肽段，尤其在鉴定含组氨酸肽段时具有明显优势。本研究建立的在线pH梯度洗脱的强阳离子交换液相色谱-质谱联用方法可以作为与反相液相色谱-质谱方法互补的一种方法，能够应用于标准蛋白、细胞总蛋白以及膜蛋白的分析中。