Native Proteome Research Articles

Determining the 3D Topologies of Heteromeric Protein Assemblies by a Mass-Spectrometry Based Hybrid Approach

Describing, understanding, and modulating the function of the cell require elucidation of the networks and structure of its macromolecular assemblies. Here, we describe an integrative approach to determine the topologies of heteromeric assemblies using structural information derived from native mass spectrometry (MS), proteomics and chemical cross-linking MS. The method was developed and further assessed for robustness and accuracy using a benchmark of five hypothetical assemblies with simulated data and two assemblies with previously published MS data. With this benchmark in hand, we purify and characterize the yeast eukaryotic initiation factor 3 (eIF3) complex whose complete 3D topology has not been previously defined. Using a combination of MS-based data, we establish sub-stoichiometric binding of eIF5 and derive a high-likelihood structural model for the five subunit eIF3 complex. Our results further reveal two interaction modules within the eIF3 and are in accord with its role as a scaffold for other initiation factors. In conclusion, here we highlight the utility of a MS-based integrative approach for modelling complexes with unknown interactions and topology. The corresponding computational algorithm is implemented in the open source Integrative Modeling Platform (IMP).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Native Protein Complexes in the Cytoplasm of Red Blood Cells

Despite decades of advancements, the investigation of the red blood cell (RBC) cytosolic proteome still represents a challenging task because of the overwhelming abundance of hemoglobin. Besides, the separation method is one of the main limiting factors when investigating protein complexes. In this study, we performed for the first time a 2D-clear native (CN)-SDS-PAGE followed by mass spectrometry-based identification to screen multiprotein complexes (MCPs) in the cytosol of human RBCs. Upstream to 2D-CN-SDS-PAGE, we applied a recently developed native pre-enrichment strategy that allows discriminating and separately collecting three distinct fractions, one of which is highly enriched for hemoglobin. Such prefractionation strategy is conservative, in that it makes soluble native-complex analyses amenable without loss of biological information. Because of the resolution of native gel electrophoresis techniques, we could observe and describe 55 potential hetero-oligomeric MPCs from the RBC native cytosolic proteome, among which ultratetrameric hemoglobin. The detected protein complexes were characterized by proteins mainly involved in oxygen transport, antioxidant responses, metabolism, and protein degradation cascades, in agreement with recent in silico models. Metabolic enzyme oligomers also interacted with complexes of proteins involved in oxidative stress responses, thus suggesting a functional relationship between metabolic modulation and antioxidant defenses.

A Chemical Proteomics Approach to Profiling the ATP-binding Proteome of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics.

Abstract 5167: Profiling GTPases in native proteomes with a GTP acyl phosphate probe.

Abstract GTPases constitute an important class of regulatory proteins that participate as signal transducers in the capacity of molecular switches. The GTPase superfamily consists of 250 members in the human genome. These fall into several families, including the small GTPases (Ras Rho, Arf, Rab and Ran), heterotrimeric GTPases, elongation and initiation factors, and dynamins. In order to profile the distribution of GTPases in various cell lines and tissues, we have developed a chemical probe comprised of GTP linked to desthiobiotin through an acyl-phosphate bond. By treating complex proteomes with the probe, we are able to isolate probe-labeled peptides with streptavidin, and subsequently, identify the probe-labeled peptides by mass spectrometry. GTPases were identified from almost all known GTPase families indicating that this approach can be used to globally profile GTPases in various proteomes. The most common labeling site occurs at the conserved lysine residue in the phosphate binding loop (GXXXXGK) of the GTP-binding site. Significantly, the probe-labeled peptide for H-,K- and N-RAS includes amino acid positions 12 and 13, which are frequently mutated in cancers. Thus, the GTP probe can be used to directly quantify mutant RAS isoforms at the protein level. Citation Format: Tyzoon K. Nomanbhoy, Ann Shih, Arwin Aban. Profiling GTPases in native proteomes with a GTP acyl phosphate probe. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2013-5167

MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae

Bacterial aminopeptidases play important roles in pathogenesis by providing a source of amino acids from exogenous proteins, destroying host immunological effector peptides and executing posttranslational modification of bacterial and host proteins. We show that MHJ_0125 from the swine respiratory pathogen Mycoplasma hyopneumoniae represents a new member of the M42 class of bacterial aminopeptidases. Despite lacking a recognizable signal sequence, MHJ_0125 is detectable on the cell surface by fluorescence microscopy and LC-MS/MS of (i) biotinylated surface proteins captured by avidin chromatography and (ii) peptides released by mild trypsin shaving. Furthermore, surface-associated glutamyl aminopeptidase activity was detected by incubation of live M. hyopneumoniae cells with the diagnostic substrate H-Glu-AMC. MHJ_0125 moonlights as a multifunctional adhesin, binding to both heparin and plasminogen. Native proteomics and comparative modelling studies suggest MHJ_0125 forms a dodecameric, homopolymeric structure and provide insight into the positions of key residues that are predicted to interact with heparin and plasminogen. MHJ_0125 is the first aminopeptidase shown to both bind plasminogen and facilitate its activation by tissue plasminogen activator. Plasmin cleaves host extracellular matrix proteins and activates matrix metalloproteases, generating peptide substrates for MHJ_0125 and a source of amino acids for growth of M. hyopneumoniae. This unique interaction represents a new paradigm in microbial pathogenesis.

Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates

The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Caspase-2 is considered an initiator caspase because its long prodomain contains a CARD domain that allows its recruitment and activation in several complexes by homotypic death domain-fold interactions. Because little is known about the function and specificity of caspase-2 and its physiological substrates, we compared the cleavage specificity profile of recombinant human caspase-2 with those of caspase-3 and -7 by analyzing cell lysates using N-terminal COmbined FRActional DIagonal Chromatography (COFRADIC). Substrate analysis of the 68 cleavage sites identified in 61 proteins revealed that the protease specificities of human caspases-2, -3, and -7 largely overlap, revealing the DEVD↓G consensus cleavage sequence. We confirmed that Asp(563) in eukaryotic translation initiation factor 4B (eIF4B) is a cleavage site preferred by caspase-2 not only in COFRADIC setup but also upon co-expression in HEK 293T cells. These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex.

The Frataxin Homologue Fra Plays a Key Role in Intracellular Iron Channeling in Bacillus subtilis

Frataxin homologues are important iron chaperones in eukarya and prokarya. Using a native proteomics approach we were able to identify the structural frataxin homologue Fra (formerly YdhG) of Bacillus subtilis and to quantify its native iron-binding stoichiometry. Using recombinant proteins we could show in vitro that Fra is able to transfer iron onto the B. subtilis SUF system for iron-sulfur cluster biosynthesis. In a four-constituents reconstitution system (including SufU, SufS, Fra and CitB) we observed a Fra-dependent formation of a [4 Fe-4 S] cluster on SufU that could be efficiently transferred onto the target apo-aconitase (CitB). A Δfra deletion mutant showed a severe growth phenotype associated with a broadly disturbed iron homeostasis; this indicates that Fra is a central component of intracellular iron channeling in B. subtilis.

In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and invivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

Folding Proteome of Yarrowia lipolytica Targeting with Uracil Permease Mutants

The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.

Coupling protein complex analysis to peptide based proteomics

Proteolysis is a central component of most proteomics methods. Unfortunately much of the information relating to the structural diversity of proteins is lost during digestion. This paper describes a method in which the native proteome of yeast was subjected to preliminary fractionation by size exclusion chromatography (SEC) prior to trypsin digestion of SEC fractions and reversed phase chromatography-mass spectral analysis to identify tryptic peptides thus generated. Through this approach proteins associated with other proteins in high molecular mass complexes were recognized and identified. A focus of this work was on the identification of Hub proteins that associate with multiple interaction partners. A critical component of this strategy is to choose methods and conditions that maximize retention of native structure during the various stages of analysis prior to proteolysis, especially during cell lysis. Maximum survival of protein complexes during lysis was obtained with the French press and bead-beater methods of cell disruption at approximately pH 8 with 200 mM NaCl in the lysis buffer. Structure retention was favored by higher ionic strength, suggesting that hydrophobic effects are important in maintaining the structure of protein complexes. Recovery of protein complexes declined substantially with storage at any temperature, but storage at −20 °C was best when low temperature storage was necessary. Slightly lower recovery was obtained with storage at −80 °C while lowest recovery was achieved at 4 °C. It was concluded that initial fractionation of native proteins in cell lysates by SEC prior to RPC-MS/MS of tryptic digests can be used to recognize and identify proteins in complexes along with their interaction partners in known protein complexes.

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

Assessing the use of thermal treatment to preserve the intact proteomes of post‐mortem heart and brain tissue

Protein degradation that occurs in tissue during post-mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap-freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2-DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high-molecular-weight proteins and reduction in protein fragmentation.

Novel Multiprotein Complexes Identified in the Hyperthermophilic Archaeon Pyrococcus furiosus by Non-denaturing Fractionation of the Native Proteome

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8-6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72-5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.

Strategies for Discovery and Characterization of Novel Biomarkers Using SELDI Technology

Successful biomarker discovery and development efforts require a working knowledge of multiple disciplines, including study and experimental design, proteomics technologies, and data analysis and interpretation. There are two general proteomic approaches to biomarker discovery: top-down and bottom-up methodologies. In bottom-up methods, complex samples are digested with a protease (typically trypsin) and subjected to an LC separation prior to analysis in a tandem mass spectrometer. In contrast, Bio-Rad's biomarker workflow focuses on native proteins and peptides top-down proteomics. This approach preserves information about post-translational modifications (truncation, glycosylation, etc.) that may be important indications of disease. Bio-Rad's Biomarker Research Center has years of expertise in addressing and circumventing common issues associated with biomarker attrition through their collaborative research services. SELDI-based biomarker studies can typically be divided into four phases: Discovery, Validation, Purification and Identification, and Assay Development, each of which requires a unique approach. The discovery phase is characterized by analyzing samples under a large set of experimental profiling conditions. An initial panel of candidate biomarkers is obtained, which is then tested during the validation phase. In the discovery and validation phases, it is especially important to optimize study design and statistical methods to avoid pre-analytical bias and yield robust markers. Identification can be performed at the end of the discovery phase or at any point during the validation phase, and generally requires standard protein purification procedures (column chromatography, size filtration, SDS-PAGE, etc.), followed by protease digestion and sequence analysis on a tandem mass spectrometer. Identification of the biomarkers facilitates the development of analyte specific assays and provides insight into the disease biology. Once the biomarkers have been positively identified, quantitati ve assays doi:10.4172/jpb.s1000200 Journal of Proteomics & Bioinformatics Open Access www.omicsonline.com Abstract No.329 JPB/Vol.S2/July 2008/Special Issue Proceedings of The Joint 2 Pacific Rim International Conference on Protein Science and 4 Asian-Oceania Human Proteome Organization, CairnsAustralia, 22-26 June 2008 J Proteomics Bioinform Volume S2:280-281(2008) ISSN:0974-276X JPB, an open access journal can be developed for routine testing on an appropriate immunoassay platform. SELDI-based immunoassays are particularly useful for detecting biomarkers with post-translational modifications. Approaches to optimizing biomarker workflows to deliver robust biomarkers and biomarker assays will be discussed using examples from Biomarker Research Center collaborations.

NK Cell Protease Granzyme M Targets α-Tubulin and Disorganizes the Microtubule Network

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

Non-disruptive release of Pseudomonas putida proteins by in situ electric breakdown of intact cells

Analysis of the native proteome of bacterial cells typically involves physical procedures (sonication, French press) and/or biochemical methods (treatment with lysozyme, osmotic shock etc.) to break open the bacteria to yield a soluble protein fraction. Such procedures are not only time consuming, but they change bacterial physiology during manipulation and affect labile post-translational modifications such as His–P bonds. In this work, we document the efficacy of the dielectric breakdown of live bacteria for releasing and delivering the protein contents of intact cells directly into a non-denaturing gel system. By means of such an in situ electrophoresis, the protein pool enters the separation medium without any manipulation of the cells other than being exposed to a moderate electric voltage. To validate the method we have followed the fate of the two forms of the PtsN (EIIA Ntr) protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) of Pseudomonas putida through the various stages of the procedure. Apart of detecting the corresponding polypeptides, we show that this procedure releases the bulk of the proteome while keeping unharmed the phosphorylation state of EIIA Ntr as it was present in the cells prior to applying the electric field. The method is applicable to other bacteria as well.

Growth-dependent Phosphorylation of the PtsN (EIINtr) Protein of Pseudomonas putida

The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EIINtr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EIINtr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN approximately P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EIINtr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His68 was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.

Activity-based probes for proteomic profiling of histone deacetylase complexes

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.