Histone deacetylases (HDACs) are key regulators of gene expression that require 
assembly into larger protein complexes for activity. Efforts to understand how 
associated proteins modulate the function of HDACs would benefit from new 
technologies that evaluate HDAC activity in native biological systems. Here, we 
describe an active site-directed chemical probe for profiling HDACs in native 
proteomes and live cells. This probe, designated SAHA-BPyne, contains structural 
elements of the general HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), 
as well as benzophenone and alkyne moieties to effect covalent modification and 
enrichment of HDACs, respectively. Both class I and II HDACs were identified as 
specific targets of SAHA-BPyne in proteomes. Interestingly, multiple 
HDAC-associated proteins were also enriched by SAHA-BPyne, even after 
denaturation of probe-labeled proteomes. These data indicate that certain 
HDAC-associated proteins are directly modified by SAHA-BPyne, placing them in 
close proximity to HDAC active sites where they would be primed to regulate 
substrate recognition and activity. We further show that SAHA-BPyne can be used 
to measure differences in HDAC content and complex assembly in human disease 
models. This chemical proteomics probe should thus prove valuable for profiling 
both the activity state of HDACs and the binding proteins that regulate their 
function.