Cell-Wide Survey of Amide-Bonded Lysine Modifications by Using Deacetylase CobB

Wan-Jie Yang,Qi-Jun Wang,Xia-Di He,Shi-Min Zhao,Peng-Cheng Lin,Yun Wei,Jian-Yuan Zhao,Wei Xu

doi:10.1186/s12575-019-0109-x

Abstract

BackgroundLysine post-translational modifications are important regulators of protein function. Proteomic and biochemical approaches have resulted in identification of several lysine modifications, including acetylation, crotonylation, and succinylation. Here, we developed an approach for surveying amide-bonded lysine modifications in the proteome of human tissues/cells based on the observation that many lysine modifications are amide-bonded and that the Salmonella enterica deacetylase, CobB, is an amidase.ResultsAfter the proteome of human tissues/cells was denatured and the non-covalently bonded metabolites were removed by acetone washes, and the amide-bonded modifiers were released by CobB and analyzed using liquid- and/or gas chromatography/mass spectrometry metabolomic analysis. This protocol, which required 3–4 days for completion, was used to qualitatively identify more than 40 documented and unreported lysine modifications from the human proteome and to quantitatively analyze dynamic changes in targeted amide-bonded lysine modifications.ConclusionsWe developed a method that was capable of monitoring and quantifying amide-bonded lysine modifications in cells of different origins.

Highlights

Lysine post-translational modifications are important regulators of protein function
While investigating the activities of Salmonella enterica deacetylase CobB [10], we unexpectedly observed that CobB possesses nonspecific amidase activity
Proteins in cell lysates were precipitated and washed extensively with acetone to remove any small molecules that were noncovalently bound to the proteins, the amide-bonded modifications to the proteome were released by CobB treatment, and the released modifiers were analyzed by liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/mass spectrometry (GC/MS)

Summary

Introduction

Lysine post-translational modifications are important regulators of protein function. Proteomic and biochemical approaches have resulted in identification of several lysine modifications, including acetylation, crotonylation, and succinylation. We developed an approach for surveying amide-bonded lysine modifications in the proteome of human tissues/cells based on the observation that many lysine modifications are amide-bonded and that the Salmonella enterica deacetylase, CobB, is an amidase. Many amino acids are modified post translationally to regulate functions of proteins. Other amino acid residues such as histidine, proline and cysteine can be phosphorylated [3, 4], hydroxylated [5] or acylated [6], respectively, to convey various biological functions. Lysine acetylation of histones and other nuclear proteins is critical for chromatin remodeling and regulation of gene tran-

Methods

Results

Discussion

Conclusion