Department of Neuro- and Sensory Physiology & Center for Biostructural Imaging of Neurodegeneration (BIN), Institut für Neuro- und Sinnesphysiologie, University Medical Center Göttingen, Göttingen, Germany.
Bioanalytical Mass Spectrometry Group, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany.
Methods Mol Biol. 2021;2218:291-302. doi: 10.1007/978-1-0716-0970-5_23.
Protein production and degradation are tightly regulated to prevent cellular structures from accumulating damage and to allow their correct functioning. A key aspect of this regulation is the protein half-life, corresponding to the time in which half of a specific protein population is exchanged with respect to its initial state. Proteome-wide techniques to investigate protein half-lives in vivo are emerging. Recently, we have established and thoroughly tested a metabolic labeling approach using C lysine (Lys(6)) for measuring protein lifetimes in mice. The approach is based on the fact that different proteins will incorporate a metabolic label at a rate that is dependent on their half-life. Using amino acid pool modeling and mass spectrometry, it is possible to measure the fraction of newly synthesized proteins and determine protein half-lives. In this chapter, we show how to extend this approach to zebrafish (Danio rerio), using a commercially available fish diet based on the stable isotope labeling by amino acids in cell culture (SILAC) technology. We describe the methods for labeling animals and subsequently use mass spectrometry to determine the lifetimes of a large number of proteins. In the mass spectrometry workflow proposed here, we have implemented the BoxCar data acquisition approach for increasing sample coverage and optimize machine use. To establish the proteome library used in the BoxCar approach, we recommend performing an in-solution digestion followed by peptide fractionation through basic reversed-phase chromatography. Overall, this chapter extends the use of current proteome technologies for the quantification of protein turnover to zebrafish and similar organisms and permits the study of germline changes following specific manipulations.
蛋白质的合成与降解受到严格调控，以防止细胞结构累积损伤，并确保其正常运作。这种调控的一个关键方面是蛋白质半衰期，即特定蛋白质群体中一半物质相对于初始状态发生交换所需的时间。目前已经出现了用于在体内研究蛋白质半衰期的蛋白质组学技术。最近，我们建立并彻底测试了一种使用 C 赖氨酸（Lys（6））的代谢标记方法，用于测量小鼠体内蛋白质的寿命。该方法基于这样一个事实，即不同的蛋白质以依赖于其半衰期的速度掺入代谢标记物。通过氨基酸池建模和质谱分析，可以测量新合成蛋白质的分数，并确定蛋白质半衰期。在本章中，我们展示了如何将这种方法扩展到斑马鱼（Danio rerio），使用基于细胞培养稳定同位素标记的氨基酸（SILAC）技术的商业可获得的鱼饲料。我们描述了标记动物的方法，然后使用质谱法来确定大量蛋白质的寿命。在我们提出的质谱工作流程中，我们采用了 BoxCar 数据采集方法来增加样本覆盖率并优化机器使用。为了建立 BoxCar 方法中使用的蛋白质组文库，我们建议进行溶液内消化，然后通过碱性反相色谱进行肽分级。总的来说，本章将当前的蛋白质组技术用于定量蛋白质周转扩展到了斑马鱼和类似的生物，并允许对特定操作后生殖系变化进行研究。
