基于蛋白质突变体和同位素标记技术的双重内标法棕榈酰化蛋白质组定量分析

Quantitative analysis of palmitoylated proteomics is of physiological and clinical significance. To date, there are only two methods reported for quantitative analysis of protein palmitoylation. One is semi-quantitative method depending on mass spectrometry (MS)-based spectral counting. The other is relative protein quantification based on stable isotope labeling of cells. We here propose a novel strategy for absolute quantitative analysis of palmitoylated proteins for the first time. Cells expressing mutant protein were metabolically labeled by 17-octadecynoic acid (17-ODYA). During the separation of palmitoylated proteins from unpalmitoylated proteins, isolated total mutant proteins were used as internal standards together with the followed isotope labeling for MS analysis. The degree of palmitoylation of target protein is described by the ratio of palmitoylated target protein labeled by heavy isotope to unpalmitoylated target protein labeled by light isotope, and accurately quantified by comparing with that of mutant protein. The absolute content of palmitoylated protein is also derived when we multiply the degree of target protein palmitoylation by its total content.The construction of internal standard breaks through the bottleneck of MS-based absolute quantitative analysis of palimitoylated proteomics. In combination with relative quantitative technique based on stable isotope labeling, the error caused by different ionization efficiencies of peptides is eliminated, although the error is common in traditional MS-based absolute quantitative analysis only using certain peptides as internal standard. The strategy described here can be extended to absolute quantitative analysis of other types of post-translational modification of protein, which will provide direct evidences for the elucidation of molecular mechanism underlying protein post-translational modification-related diseases.

本项目首次提出了一种基于质谱的棕榈酰化蛋白质组绝对定量分析的全新策略：以人工基因表达合成的蛋白质突变体和17－十八炔酸（17-ODYA）代谢标记的该蛋白质突变体作为"内标"，结合同位素标记相对定量技术，通过比较目标蛋白与"内标"蛋白在棕榈酰化蛋白质组（"重"同位素标记）和未棕榈酰化蛋白质组（"轻"同位素标记）的相对定量关系，实现活细胞中目标蛋白质棕榈酰化率（棕榈酰化蛋白质的量/未棕榈酰化蛋白质的量）的精确定量及棕榈酰化蛋白质绝对含量的测定。目前已见诸报道的棕榈酰化蛋白质组定量分析方法多系半定量或相对定量分析，本项目中"内标"的构建突破了质谱法棕榈酰化蛋白质组绝对定量分析的瓶颈。将"内标"法和同位素标记相对定量技术结合，避免了传统内标法绝对定量分析时因肽段的离子化效率不同而产生的误差。该策略可拓展至其它蛋白质翻译后修饰蛋白质组的绝对定量分析，对与之相关的疾病的诊断和治疗具有明显的现实意义。

蛋白质棕榈酰化修饰是蛋白质翻译后修饰的重要形式之一，在细胞内蛋白质转运、细胞定位、以及细胞膜相关信号转导等方面起着关键的作用。棕榈酰化修饰蛋白质的精确定量分析对于理解该修饰形式的生物学和生理学意义至关重要。本项目将稳定同位素标记技术和高分辨质谱分析技术相结合，建立了一种活细胞中蛋白质棕榈酰化率精确定量的分析方法。目前已报道的棕榈酰化蛋白质组的定量方法仅仅针对棕榈酰化修饰后的蛋白质进行分析，忽略了蛋白质自身合成量的变化。本项目建立的分析方法通过对蛋白质棕榈酰化率（即棕榈酰化蛋白质的量/总蛋白质的量）的精确定量，更能准确地描述蛋白质棕榈酰化修饰的过程，更有利于直观地判断与蛋白质棕榈酰化修饰相关的疾病的发生和发展，对于临床疾病的诊断和治疗具有明显的现实意义。本项目将建立的蛋白质棕榈酰化率精确定量的分析方法应用于Traf6敲减对人结肠癌细胞SW480细胞内棕榈酰化蛋白质表达量影响的研究以验证本方法的有效性和可行性。结果发现，Traf6在调控棕榈酰化蛋白表达量的同时，亦影响了相应自身蛋白的合成和表达。因此，相比较针对于棕榈酰化蛋白的直接定量分析，目标蛋白质棕榈酰化率的精确定量分析提供的生物学信息具有更高的可信度。同时，本项目将建立的蛋白质棕榈酰化率精确定量的分析方法应用于脂肪酸2-羟基化酶对小窝蛋白caveolin-1棕榈酰化率的影响，发现脂肪酸2-羟基化酶的过表达降低了了caveolin-1的棕榈酰化率，说明脂肪酸2-羟基化酶的过表达抑制了caveolin-1的棕榈酰化修饰过程，通过western blot实验对上述结论进行了生物验证。