基于液相色谱质谱联用的蛋白质组非标记定量研究策略的建立及应用

The Evaluation and Application of a Label-free Quantitative Proteomic Strategy Based on LC-ESI-MS/MS

定量蛋白质组研究是蛋白质组研究的热点和难点,而液相色谱质谱技术已经被广泛地应用于蛋白质的定性和定量研究.该研究建立和优化了一种基于液相色谱质谱联用技术的蛋白质组非标记定量方法,并对两种肽段质谱检测计数的归一化算法进行了比较,结果发现ASC法要优于RSC法.最后,将建立的方法应用于肝癌细胞模型HepG2和HepG2-HBx细胞系的差异蛋白质组表达研究.质谱鉴定结果用聚类分析软件Cluster3.0进行分析,最后鉴定出107个重叠蛋白,其中9个蛋白质表达上调(Ratio>1.75),6个蛋白质表达下调(Ratio<0.5),这些蛋白质均与肝癌发生和恶化密切相关.结果表明,该技术操作简单、方便,具有较高的灵敏度和动态范围,利用该方法进行差异蛋白质组研究和发现生物标志物在理论和临床上具有十分重要的意义.

Mass spectrometry is being widely applied to identify and quantify proteins in complex mixtures. Quantification of small molecules by integration of LC-MS extracted ion chromatogram （XIC） peaks has a long history in analytical chemistry. Similar quantification techniques applied to proteolytic protein digests have also been previously described. A comprehensive approach for label-free quantification using yeast proteome as a model have been developed. Based on spectra counts of peptides, the relative protein quantification from LC-MS/MS experiments of proteolytic protein digests was performed. Unlabeled protein samples were digested with trypsin and separated by one-dimensional nano-flow HPLC （RPLC）, and mass spectra were obtained by using the survey mode of LTQ mass spectrometer with dynamic exclusion. The correlating relationship between concentrate of protein and spectra counts was confirmed. Two algorithms to normalize spectra counts ratio from different samples were compared, and the results suggested that algorithm based on average spectra count （ASC） was eximious. The method was used to biomarkers discovery in HepG2 and HepG2-HBx cell lines. The identified proteins were analyzed and classified by cluster software （Version 3.0）. Finally, 107 overlap proteins were identified, among them, 9 proteins were identified up-regulated （Ratio 〉 1.75） and 6 proteins down-regulated （Ratio 〈 0.5）. Further research indicated that these proteins were related with liver cancer. Altogether, the results indicated that the strategy was operable and convenient with high sensitivity and wild dynamic range, and will be significant to deliver biomarker discovery either in theory or in clinic.