Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards
Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards
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DOI：
10.1021/ac026154
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发表时间：
2003-02-01
影响因子：
7.4
通讯作者：
Lindall, A
中科院分区：
文献类型：
--
作者：
Barnidge, DR;Dratz, EA;Lindall, A
描述了使用同位素标记的或未标记的合成肽作为标准品对膜蛋白进行绝对定量的方法。设计合成肽以模拟通过蛋白水解或化学消化从靶分析物蛋白质裂解的肽，并且所选肽用作在三重四极杆质谱仪上通过LC/MS/MS定量的标准品。该技术是对广泛使用的相对定量技术的补充，通过提供具有更高灵敏度、动态范围和精确度的所选靶标的绝对定量。通过全局蛋白质组搜索发现的感兴趣的蛋白质可以被选择作为通过本方法定量的靶。这种方法具有更短的分析周期时间（对于全局蛋白质组实验，几分钟相对于几小时），使其非常适合高通量环境。本方法使用合成肽作为标准品，结合靶蛋白的蛋白水解或化学裂解，允许质谱法用作高度选择性的检测器，用于提供没有标准品可用的蛋白质的绝对定量。我们证明，定量是简单可靠的，为完整的膜蛋白视紫红质与合理的回收率重复实验使用低微摩尔溶液视紫红质杆外段。
Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.