Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis
The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after “pulse” labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein’s half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo.
Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide.
Key wordsProteomics Mass spectrometry Protein half-life Protein decay dynamics Stable isotope labeling of mammals Nitrogen-15 SILAC SILAM Extremely long-lived proteins
Funding for JRY has been provided by National Institutes of Health grants P41 GM103533, R01 MH067880, R01 MH100175, UCLA/NHLBI Proteomics Centers (HHSN268201000035C). JNS is supported by the Pathway to Independence Award National Institutes of Health (K99DC013805). We acknowledge Martin Hetzer and Brandon Toyama for their involvement in initializing the project and also Varda Levram-Ellisman and Roger Tsien for their input.
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