Konopka Anna, Zinn Nico, Wild Christina, Lehmann Wolf D
Molecular Structure Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, Heidelberg, 69120, Germany.
Methods Mol Biol. 2014;1156:337-63. doi: 10.1007/978-1-4939-0685-7_23.
A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy.
定量蛋白质组学进一步发展的一个主要障碍是缺乏准确量化的蛋白质标准品。以下方案描述了生产稳定同位素标记蛋白质标准品的创新方法。它们通过无细胞蛋白质合成来生产，这种方法能够同时掺入硒代甲硫氨酸和稳定同位素标记的氨基酸。硒标签允许通过电感耦合等离子体质谱（ICP-MS）进行灵敏且准确的定量。稳定同位素标记允许在基于质谱的蛋白质组学中通过电喷雾电离串联质谱（ESI-MS/MS）进行内标标准化。两种标记类型可以置于单个蛋白质RISQ标准品（重组同位素标记且硒定量）中，或者可以分布在针对同一目标蛋白质的两种相关RSQ和RIQ标准品上（重组硒定量以及重组同位素标记且定量）。将无细胞合成作为生产方法与ICP-MS和ESI-MS/MS作为检测方法相结合，可得到具有极高准确度的蛋白质标准品。
