使用DO代谢标记和混合液相色谱-质谱法测量小鼠肝脏中HSD17β13蛋白的周转。
Measuring HSD17β13 protein turnover in mouse liver with DO metabolic labeling and hybrid LC-MS.
作者信息
Shi Yifan, Del Rosario Amanda, Wang Sheng-Ping, Kang Lijuan, Liu Haiying, Rady Brian, Jian Wenying
机构信息
Janssen Research & Development, Spring House, PA, USA.
出版信息
Bioanalysis. 2025 Feb;17(3):151-159. doi: 10.1080/17576180.2025.2452757. Epub 2025 Jan 17.
BACKGROUND
Metabolic labeling with heavy water (DO) followed by LC-MS has become a powerful tool for studying protein turnover . 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 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 using DO metabolic labeling.
背景
用重水（D₂O）进行代谢标记后结合液相色谱-质谱联用（LC-MS）已成为研究蛋白质周转的有力工具。开发一种定量方法来测量部分标记的低丰度蛋白质面临诸多挑战，因为肽段的重同位素异构体，尤其是它们通过氘标记后的变化，很难检测。
方法
提出了一种将免疫捕获与液相色谱-高分辨率质谱联用的工作流程，以测定小鼠肝脏中HSD17β13蛋白的合成速率。分析了胰蛋白酶肽段的氘标记情况，并将数据拟合到指数上升方程中。
结果与结论
从3种不同的肽段计算出HSD17β13蛋白的半衰期分别为31.8、36.1和28.9小时，平均为32.3小时。所建立的工作流程可从混合LC-MS蛋白质定量分析方法改编而来，用于使用D₂O代谢标记评估蛋白质周转。
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