RESUMO
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 13C 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.
