Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay.
Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay.
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DOI：
10.21769/bioprotoc.4873
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发表时间：
2023-11-05
期刊：
影响因子：
0.8
通讯作者：
Bai, Hua
中科院分区：
文献类型：
--
作者：
Miao, Ting;Bai, Hua
关键词：
赖氨酸乙酰化是一种保守的翻译后修饰，是多种细胞过程的关键调控机制，包括代谢控制、表观遗传调控和细胞信号转导。质谱（MS）的最新进展使得广泛鉴定组蛋白和非组蛋白的乙酰化赖氨酸残基成为可能。然而，在质谱分析之前进行蛋白质富集可能是必要的，以提高对低丰度蛋白质或表现出低乙酰化水平的蛋白质的检测。脂肪酸合成酶（FASN）是一种催化脂肪酸从头合成的必需酶，在从果蝇到人类的各种物种中都被发现是乙酰化的。在这里，我们描述了一个基于抗体的蛋白质富集和样品制备的一步一步的过程，通过基于ms的蛋白质组学分析来鉴定内源性FASN蛋白的乙酰化。同时，我们提供了一种用于FASN活性测量的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）吸光度测定的方案，FASN活性是新生脂肪生成的主要功能读数之一。•一个全面的协议蛋白质免疫沉淀和样品制备乙酰化位点鉴定的质谱。•使用吸光度法逐步测量果蝇幼虫FASN活性的程序。
Lysine acetylation is a conserved post-translational modification and a key regulatory mechanism for various cellular processes, including metabolic control, epigenetic regulation, and cellular signaling transduction. Recent advances in mass spectrometry (MS) enable the extensive identification of acetylated lysine residues of histone and non-histone proteins. However, protein enrichment before MS analysis may be necessary to improve the detection of low-abundant proteins or proteins that exhibit low acetylation levels. Fatty acid synthase (FASN), an essential enzyme catalyzing the de novo synthesis of fatty acids, has been found to be acetylated in various species, from fruit flies to humans. Here, we describe a step-by-step process of antibody-based protein enrichment and sample preparation for acetylation identification of endogenous FASN protein by MS-based proteomics analysis. Meanwhile, we provide a protocol for nicotinamide adenine dinucleotide phosphate (NADPH) absorbance assay for FASN activity measurement, which is one of the primary functional readouts of de novo lipogenesis. Key features • A comprehensive protocol for protein immunoprecipitation and sample preparation for acetylation site identification by mass spectrometry. • Step-by-step procedures for measurement of FASN activity of fruit fly larvae using an absorbance assay.