Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry
Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry
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DOI：
10.1021/ac034607k
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发表时间：
2003-12-01
影响因子：
7.4
通讯作者：
Shen, RF
中科院分区：
文献类型：
--
作者：
Chelius, D;Zhang, T;Shen, RF
一个简单而可靠的方法，这里描述了复杂的混合物中的蛋白质的识别和相对定量使用二维液相色谱/串联。质谱分析法来该方法基于经典的蛋白质组学分析，其中蛋白质用胰蛋白酶消化，并且所得肽通过多维液相色谱法分离。通过串联质谱法分析分离的肽，并通过数据库搜索算法如SEQUEST进行鉴定。计算所有鉴定的肽的峰面积（肽洗脱时间内的积分离子计数），并通过比较一个样品中该蛋白质的所有肽的峰面积与另一个样品中该蛋白质的所有肽的峰面积来确定每种蛋白质的相对浓度。使用这种策略，我们比较了在表皮生长因子（EGF）存在或不存在下生长的A431细胞（表皮细胞系）的蛋白质表达的相对水平。我们的研究结果与发表的观察EGF的瞬态效应是一致的。此外，在我们的研究中确定的几个磷酸肽的浓度的差异表明参与EGF细胞信号通路的几个新的目标的可能性。这种全球性的蛋白质鉴定和定量技术应该被证明是一种有价值的手段，用于比较生物样品中的蛋白质组进行差异化处理。
A simple and reliable method is described here for the identification and relative quantification of proteins in complex mixtures using two-dimensional liquid chromatography/tandem. mass spectrometry. The method is based on the classical proteomic analysis where proteins are digested with trypsin and the resulting peptides are separated by multidimensional liquid chromatography. The separated peptides are analyzed by tandem mass spectrometry and identified via a database search algorithm such as SEQUEST. The peak areas (integrated ion counts over the peptide elution time) of all identified peptides are calculated, and the relative concentration of each protein is determined by comparing the peak areas of all peptides from that protein in one sample versus those from the other. Using this strategy, we compared the relative level of protein expression of A431 cells (an epidermal cell line) grown in the presence or absence of epidermal growth factor (EGF). Our results are consistent with the published observations of the transient effects of EGF. In addition, the difference in the concentrations of several phosphopeptides determined in our studies suggests the possibility of several new targets involved in the EGF cell-signaling pathway. This global protein identification and quantification technology should prove to be a valuable means for comparing proteomes in biological samples subjected to differential treatments.