Measuring HSD17β13 protein turnover in mouse liver with D(2)O metabolic labeling and hybrid LC-MS.

BACKGROUND: Metabolic labeling with heavy water (D(2)O) followed by LC-MS has become a powerful tool for studying protein turnover in vivo. Developing a quantitative method to measure partially labeled low-abundance proteins poses many challenges because heavy isotopomers of peptides, especially their changes through deuterium labeling, are difficult to detect. METHODS: A workflow that coupled immunocapture and LC-high-resolution MS to determine the synthesis rate of HSD17β13 protein in mouse liver was presented. Deuterium labeling of tryptic peptides was analyzed, and data were fitted into an exponential rise equation. RESULTS & CONCLUSION: HSD17β13 protein t(1/2) were calculated to be 31.8, 36.1, and 28.9 hr from 3 different peptides with an average of 32.3 hr. The established workflow can be adapted from hybrid LC-MS protein quantitation assays to assess protein turnover in vivo using D(2)O metabolic labeling.