Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re-quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.

中文翻译：

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基于UV活性标签的靶向蛋白质组学的稳定同位素标记的肽标准品绝对定量的新方法。

靶向蛋白质组学取决于稳定同位素标记（SIL）肽标准品的可用性，对于绝对蛋白质定量，必须对其进行绝对定量。在本研究中，开发了三种绝对定量SIL肽的新方法。所有方法都依赖于具有特定紫外线吸收的定量标签（Qtag）。Qtag在合成过程中附着在肽上，并在标准蛋白质组学工作流程条件下通过胰蛋白酶消化除去。尽管设计了一种定量方法（方法A）以快速，经济地生产绝对定量的SIL肽，但开发了另外两种方法（方法B和C）以能够在重构后直接对SIL肽进行重新定量以控制和监测与肽溶解度，沉淀，并粘附在小瓶上。当彼此比较以及与氨基酸分析定量相比时，所有方法均产生一致的结果。精确的定量方法用于表征H3特异性组蛋白甲基转移酶EZH2的体内特异性。