D2O代谢标记和杂交LC-MS检测小鼠肝脏中HSD17β13蛋白的转换。

IF 1.9 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS Bioanalysis Pub Date : 2025-01-17 DOI:10.1080/17576180.2025.2452757

Yifan Shi, Amanda Del Rosario, Sheng-Ping Wang, Lijuan Kang, Haiying Liu, Brian Rady, Wenying Jian

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引用次数: 0

摘要

背景：用重水（D2O）进行代谢标记，然后用LC-MS进行标记，已经成为研究体内蛋白质转换的有力工具。开发一种定量方法来测量部分标记的低丰度蛋白质面临许多挑战，因为肽的重同位素体，特别是它们通过氘标记的变化，很难检测到。方法：建立免疫捕获和lc -高分辨率质谱联用测定小鼠肝脏中HSD17β13蛋白合成速率的工作流程。分析了色氨酸的氘标记，并将数据拟合为指数上升方程。结果与结论：HSD17β13蛋白t1/2分别为31.8、36.1和28.9小时，平均为32.3小时。建立的工作流程可以适应于混合LC-MS蛋白质定量分析，使用D2O代谢标记来评估体内蛋白质周转。

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Measuring HSD17β13 protein turnover in mouse liver with D₂O metabolic labeling and hybrid LC-MS.

Background: Metabolic labeling with heavy water (D₂O) 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 D₂O metabolic labeling.

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

审稿时长

2 months

期刊介绍： Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing. The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality. Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing. The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques. Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.