Vandemoortele Giel, Staes An, Gonnelli Giulia, Samyn Noortje, De Sutter Delphine, Vandermarliere Elien, Timmerman Evy, Gevaert Kris, Martens Lennart, Eyckerman Sven
VIB Medical Biotechnology Center, B-9000 Ghent, Belgium.
Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Sci Rep. 2016 Jun 6;6:27220. doi: 10.1038/srep27220.
The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins.
利用蛋白质标签来促进对目标蛋白质的详细表征，不仅给细胞生物学带来了变革，还通过有效回收含有被标记蛋白质的蛋白质复合物实现了生化分析。在能够进行高效同源重组的低等生物中，将这些标签用于内源性蛋白质的详细表征十分普遍。随着基因组工程技术的最新进展，现在大多数实验系统，包括哺乳动物细胞系培养物，都能够对内源性蛋白质进行标记。在这项工作中，我们描述了如何选择具有理想质谱特征的肽段，用于通过靶向蛋白质组学对标记蛋白质进行定量分析。我们挖掘了嗜热栖热菌的蛋白质组，以获得在所有已知模式生物的蛋白质组中都独一无二的两种肽段（蛋白质型），从而能够在复杂背景下对目标蛋白质进行灵敏定量。通过将这些“用于SRM定量的蛋白质型肽段”（PQS肽段）与表位标签相结合，我们展示了它们在转染蛋白质对后进行的免疫共沉淀实验中的应用，或者在通过基因组工程将这些标签引入内源性蛋白质后进行的免疫共沉淀实验中的应用。用于绝对定量的内源性蛋白质标记为蛋白质分析提供了一个强大的额外维度，能够对内源性蛋白质进行详细表征。
