Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay
Year: 2023
Type: article
Abstract: 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.
Source: BIO-PROTOCOL
Authors: Ting Miao, Hua Bai
Institutions: Harvard University, Iowa State University
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4388428517