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Title: Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling
Abstract

Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC.

Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin

Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization

Support Protocol 1: Expression and purification of HAT mutants

Support Protocol 2: Synthesis of Ac‐CoA surrogates

Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates

Basic Protocol 3: Chemoproteomic identification of HAT substrates

Basic Protocol 4: Validation of specific HAT substrates with western blotting

 
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Award ID(s):
1808087
PAR ID:
10381136
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
2
Issue:
7
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    Basic Protocol 1: Synthesis of the azide derivatives of chain transfer agent and radical initiator

    Basic Protocol 2: Installation of an azide group on the α‐end of RAFT polymers

    Alternate Protocol: Installation of an azide group on the ω‐end of RAFT polymers

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    Basic Protocol 1: Preparation ofArabidopsisseedling materials

    Basic Protocol 2: Isolation and enrichment of plasma membrane proteins

    Support Protocol 1: Determination of protein concentration using BCA assay

    Basic Protocol 3: Enrichment of S‐acylated proteins by acyl‐biotin exchange method

    Support Protocol 2: Protein precipitation by methanol/chloroform method

    Basic Protocol 4: Trypsin digestion and proteomic analysis

    Alternate Protocol: Pre‐resin digestion and peptide‐level enrichment

     
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