Protocol for identifying OXCT1-mediated LACTB succinylation sites in vitro

Wenhao Ma; Yuchen Sun; Huafeng Zhang

doi:10.1016/j.xpro.2024.103088

Protocol for identifying OXCT1-mediated LACTB succinylation sites in vitro

STAR Protoc. 2024 Jun 21;5(2):103088. doi: 10.1016/j.xpro.2024.103088. Epub 2024 May 23.

Authors

Wenhao Ma¹, Yuchen Sun², Huafeng Zhang³

Affiliations

¹ Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; Anhui Province Key Laboratory of Biomedical Aging Research, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China. Electronic address: wenhao99@mail.ustc.edu.cn.
² Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; Anhui Province Key Laboratory of Biomedical Aging Research, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China.
³ Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China; Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, Anhui 230601, China; Anhui Province Key Laboratory of Biomedical Aging Research, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, China. Electronic address: hzhang22@ustc.edu.cn.

Abstract

OXCT1 acts as a succinyltransferase to promote serine beta-lactamase-like protein (LACTB) K284 succinylation. Here, we present a protocol for detecting OXCT1-mediated LACTB succinylation levels and sites. We describe steps for using western blotting (WB) and mass spectrometry to determine OXCT1-mediated LACTB succinylation levels and sites in vitro. This protocol can be applied to detect and identify succinylation levels and sites on other proteins. For complete details on the use and execution of this protocol, please refer to Ma et al.¹.

Keywords: cell biology; cell-based assays; protein biochemistry.

MeSH terms

Blotting, Western / methods
Mass Spectrometry / methods
Protein Processing, Post-Translational
Succinic Acid / chemistry
Succinic Acid / metabolism
beta-Lactamases* / chemistry
beta-Lactamases* / metabolism

Substances

beta-Lactamases
Succinic Acid