The authors acknowledge COST Action CM1406 (Epigenetic Chemical Biology). Research reported in this publication was supported by the Pierre Mercier foundation.

1. Cell Culture
NOTE: The following steps are performed in a standard tissue culture hood. Familiarity with sterile technique is expected.
<ol>
	<li>Culture HeLa cells to 80% confluency in a T75 cell culture flask in MEM supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 mg/mL). 
	NOTE: Cell culture, treatment, and lysis steps are conducted under laminar flow, with cell incubation steps at 37 &#176;C performed in a humidified atmosphere of 5% CO2 under sterile conditions.</li>
	<li>Wash the cells twice with 5 mL of DPBS, aspirate the DPBS, and add 1 mL of cell dissociation reagent. Incubate at 37 &#176;C for 5 min to detach the cells.</li>
	<li>Add 9 mL of MEM culture medium, thoroughly resuspend the cell suspension, and count the cells using a hemocytometer.</li>
	<li>Adjust the density of the cell suspension to 6 x 104 cells/mL in MEM.</li>
	<li>Add 100 &#181;L of cell suspension to control and test the wells of a sterile, flat-bottom, tissue culture-treated 96-well plate (Figure 1A).</li>
	<li>Add 100 &#181;L of MEM culture medium to blank wells of the 96-well plate (Figure 1A).</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 24 h. 
	NOTE: A 24 h incubation is a suitable time to test for endogenous HDAC activity in HeLa cells. Different cell types with unknown HDAC activity may need a timecourse evaluation of control cells.</li>
</ol>
2. Cell Treatment with Test Compounds and HDAC Substrates
<ol>
	<li>Aspirate the medium from the wells of the 96-well plate.</li>
	<li>Add 25 &#181;L of 2x test compounds (e.g., trichostatin A, MS275, and tubastatin A) in MEM to test wells. Add 25 &#181;L of MEM to control and blank wells. 
	NOTE: The concentration of test compounds is to be adjusted to provide a range of at least 5 final test concentrations (one concentration per well). The DMSO final concentration in the test compound should not exceed 0.5% and should be the same in each well, including blanks and controls. The final incubation volume is 50 &#181;L per well. Trichostatin A is tested at 5 concentrations ranging from 500 to 1.95 nM (1:4 dilution). MS275 and tubastatin A are tested at 5 concentrations ranging from 8,000 to 6.17 nM (1:6 dilution). The desired final test concentration for each test compound should be determined by each individual user by creating a dose-response curve.</li>
	<li>Add 25 &#181;L of the substrate mixture (42 &#181;M of MAL, 42 &#181;M of MOCPAC, and 42 &#181;M of BATCP in MEM) to control and test wells. Add 25 &#181;L of MEM to blank wells. 
	NOTE: The final concentration of each substrate is 21 &#181;M. MOCPAC and BATCP distinguish between HDAC1 and HDAC6 activity, respectively (Figure 1B). When no activity is observed with those substrates, the use of MAL could potentially lead to the identification of activity against another HDAC isoform.</li>
	<li>Incubate the 96-well plate at 37 &#176;C for 8 h in a humidified 5% CO2 incubator. 
	NOTE: This incubation time is suitable for HeLa cells and allows the endogenous HDAC to interact with the synthetic substrates. Different cell lines may require adjustments in incubation time and/or substrate concentration.</li>
</ol>
3. Cell Lysis and Sample Preparation for UHPLC-ESI-MS/MS
Caution: The sample preparation steps use organic chemicals, which are highly flammable and toxic by ingestion or inhalation. Wear appropriate personal protection, such as gloves and safety glasses.
<ol>
	<li>Add 10 &#181;L of 6x RIPA buffer (diluted from 10x RIPA buffer) supplemented with protease inhibitor to each well of the 96-well plate.</li>
	<li>Stop the reaction by adding 160 &#181;L of cold acetonitrile to each well and mix by pipetting up and down. 
	NOTE: Keep the acetonitrile at -20 &#176;C prior to this step.</li>
	<li>Place the plate in a -80 &#176;C freezer for 10 min.</li>
	<li>Remove the plate from the freezer and transfer 220 &#181;L of the content of each well to a non-sterile, conical-bottom (V-bottom) 96-well plate. Centrifuge the plate at 5,000 x g and 4 &#176;C for 10 min. 
	NOTE: This step aims at removing proteins and salts. If necessary, the solutions can be transferred to individual conical centrifuge tubes prior to centrifugation or filtered through a set of 96-well, 0.65 &#181;m PVDF filter plates. The successful removal of the precipitate is required prior to UHPLC analysis.</li>
	<li>Transfer 200 &#181;L of supernatant (or filtrate) into a 96-well plate compatible with the UHPLC system and seal it with peelable, heat-sealing foil using a plate sealer. 
	NOTE: Avoid pipetting the pellets when removing the supernatants. If the UHPLC analysis cannot be immediately performed, the plate can be stored at 4 &#176;C for a maximum of 24 h until analysis.</li>
</ol>
4. UHPLC/MS-MS Analysis
<ol>
	<li>Prepare the UHPLC system by filling it with the mobile phase system (95% A:5% B): 
	A: H2O, HPLC-grade, containing 0.1% formic acid (prepare 1 L) 
	B: Acetonitrile, HPLC-grade, containing 0.1% formic acid (prepare 1 L). 
	Caution: The mobile phase contains organic chemicals, which are highly flammable and toxic by ingestion or inhalation. Wear appropriate personal protection, such as gloves and safety glasses.</li>
	<li>Place the 96-well plate into the sample manager containing a plate holder. Run the samples according to the UHPLC-ESI-MS/MS conditions provided in Table 1. 
	NOTE: Every column of the analytical 96-well plate should have one control well and one blank well (as depicted in the plate scheme of Figure 1A). Controls are representative of 100% enzymatic activity in the tested cell line, while blanks are there to check whether the MS source is providing consistent background. Unusual MS peaks detected in a given blank should be investigated to prevent MS sensitivity changes.</li>
</ol>
5. Data Analysis
<ol>
	<li>Integrate the peak area of each substrate and its deacetylated product with an appropriate UHPLC integration software. 
	NOTE: Use automatic integration parameters with a smoothing method (Mean, window size 3, and number of smooth 2, for example). Using the chromatographic conditions provided in Table 1, the elution order is dMAL (5.8 min), dBATCP (6.0 min), dMOCPAC (6.2 min), MAL (7.6 min), MOCPAC (8.9 min), and BATCP (9.8 min), as depicted in Figure 1C.</li>
	<li>For each substrate, calculate the peak area ratio (deacetylated/acetylated) in each chromatogram (test and control samples).</li>
	<li>Calculate the percent HDAC inhibition of each test sample: 
	<img alt="Equation" src="/files/ftp_upload/55878/55878eq1.jpg" /> 
	NOTE: HDAC1 inhibition is calculated using the peak area ratio of dMOCPAC/MOCPAC; HDAC6 inhibition is calculated with dBATCP/BATCP. The ratio dMAL/MAL can be used to express general HDAC inhibition.</li>
</ol>

<ol>
	<li>Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K., Schapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+protein+families:+a+new+frontier+for+drug+discovery.">Epigenetic protein families: a new frontier for drug discovery.</a> Nat Rev Drug Discov. 11 (5), 384-400 (2012).</li><li>Henikoff, S., Greally, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics,+cellular+memory+and+gene+regulation.">Epigenetics, cellular memory and gene regulation.</a> Curr Biol. 26 (14), R644-R648 (2016).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+inhibitor+blocks+amyloid+beta-induced+impairment+of+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 inhibitor blocks amyloid beta-induced impairment of mitochondrial transport in hippocampal neurons.</a> PLoS One. 7 (8), (2012).</li><li>Kawaguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deacetylase+HDAC6+regulates+aggresome+formation+and+cell+viability+in+response+to+misfolded+protein+stress.">The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress.</a> Cell. 115 (6), 727-738 (2003).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+of+p53+at+lysine+373/382+by+the+histone+deacetylase+inhibitor+depsipeptide+induces+expression+of+p21(Waf1/Cip1).">Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1).</a> Mol Cell Biol. 26 (7), 2782-2790 (2006).</li><li>Berger, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+in+transcriptional+regulation.">Histone modifications in transcriptional regulation.</a> Curr Opin Genet Dev. 12 (2), 142-148 (2002).</li><li>Sim&#245;es-Pires, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+as+a+target+for+neurodegenerative+diseases:+what+makes+it+different+from+the+other+HDACs?.">HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs?.</a> Mol Neurodegener. 8 (7), 1-16 (2013).</li><li>Mottamal, M., Zheng, S., Huang, T. L., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+in+clinical+studies+as+templates+for+new+anticancer+agents.">Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents.</a> Molecules. 20 (3), 3898-3941 (2015).</li><li>Manal, M., Chandrasekar, M. J., Gomathi Priya, J., Nanjan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+histone+deacetylase+as+antitumor+agents:+A+critical+review.">Inhibitors of histone deacetylase as antitumor agents: A critical review.</a> Bioorg Chem. 67, 18-42 (2016).</li><li>Mack, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+selectivity+and+beyond.">To selectivity and beyond.</a> Nat Biotech. 28 (12), 1259-1266 (2010).</li><li>. Study of ACY-1215 alone and in combination with bortezomib and dexamethasone in multiple myeloma (ACY-1215) Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01323751">https://clinicaltrials.gov/ct2/show/NCT01323751</a> (1211)</li><li>. Study of ACY-1215 in combination with lenalidomide, and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01583283">https://clinicaltrials.gov/ct2/show/NCT01583283</a> (2012)</li><li>. 13ACY-1215 (ricolinostat) in combination with pomalidomide and low-dose dex in relapsed-and-refractory multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01997840">https://clinicaltrials.gov/ct2/show/NCT01997840</a> (2013)</li><li>. Study of ACY-241 alone and in combination with pomalidomide and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT02400242">https://clinicaltrials.gov/ct2/show/NCT02400242</a> (2015)</li><li>Yee, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ricolinostat+plus+lenalidomide,+and+dexamethasone+in+relapsed+or+refractory+multiple+myeloma:+a+multicentre+phase+1b+trial.">Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial.</a> Lancet Oncol. 17 (11), 1569-1578 (2016).</li><li>Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenous+non-isotopic+assays+for+histone+deacetylase+activity.">Homogenous non-isotopic assays for histone deacetylase activity.</a> Expert Opin Ther Pat. 13 (6), 935 (2003).</li><li>Zwick, V., Sim&#245;es-Pires, C., Cuendet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+multi-substrate+assay+coupled+to+UHPLC-ESI-MS/MS+for+a+quick+identification+of+class-specific+HDAC+inhibitors.">Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors.</a> J Enzyme Inhibi Med Chem. 31 (1), 209-214 (2016).</li><li>Khan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+class+and+isoform+selectivity+of+small-molecule+histone+deacetylase+inhibitors.">Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors.</a> Biochem J. 409 (2), 581-589 (2008).</li><li>Glaser, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+acetylation+induced+by+novel+histone+deacetylase+inhibitors.">Differential protein acetylation induced by novel histone deacetylase inhibitors.</a> Biochem Biophys Res Commun. 325 (3), 683-690 (2004).</li><li>Butler, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+and+simple+chemistry+yield+a+superior,+neuroprotective+HDAC6+inhibitor,+tubastatin+A.">Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A.</a> J Am Chem Soc. 132 (31), 10842-10846 (2010).</li><li>Jung, M., Yong, K. -. J., Velena, A., Lee, S., Sippl, W., Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+for+HDAC+Inhibitor+Hit+Validation.">Cell-Based Assays for HDAC Inhibitor Hit Validation.</a> Epigenetic Targets in Drug Discovery. , (2010).</li><li>Milli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+cellular+response+to+novel+proapoptotic+agents+related+to+atypical+retinoids+in+human+IGROV-1+ovarian+carcinoma+cells.">Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.</a> J Proteome Res. 10 (3), 1191-1207 (2011).</li><li>Zwick, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+selective+HDAC6+inhibitor+active+in+neuroblasts.">Synthesis of a selective HDAC6 inhibitor active in neuroblasts.</a> Bioorg Med Chem Lett. 26 (20), 4955-4959 (2016).</li><li>Ciossek, T., Julius, H., Wieland, H., Maier, T., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homogeneous+cellular+histone+deacetylase+assay+suitable+for+compound+profiling+and+robotic+screening.">A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.</a> Anal Biochem. 372 (1), 72-81 (2008).</li><li>Heltweg, B., Dequiedt, F., Marshall, B. L., Brauch, C., Yoshida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype+selective+substrates+for+histone+deacetylases.">Subtype selective substrates for histone deacetylases.</a> J Med Chem. 47 (21), 5235-5243 (2004).</li><li>Copeland, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation of enzyme inhibitors in drug discovery. , (2005).</li></ol>

The authors have nothing to disclose.

HDAC inhibition is a hot topic in drug discovery, with a current focus on HDAC6-selective inhibitors for cancer therapy21. HDAC selectivity is usually assessed by a series of high-throughput, cell-free assays, which intend to determine the inhibitory potency towards individual HDAC isoforms21. However, the selectivity of an inhibitor must be additionally confirmed in living cells by evaluating the acetylation status of endogenous protein substrates, such as histones (for class I HDACs) and tubulin (for HDAC6). Therefore, cell assays involving antibody recognition (e.g., immunostaining, western blot, flow cytometry, and ELISA) are usually necessary to achieve a quantitative or semi-quantitative evaluation of isoform potency of a given compound in living cells21. It is not rare that a compound that was selective in a cell-free enzymatic assay turns out to be non-selective in cells or tissues17,22, and vice versa19. This can be explained by differences in non-standardized enzymatic sources and the absence of co-factors or protein partners in biochemical assays21.
The method described here is a rapid, antibody-free alternative allowing for the direct measurement of class I (HDAC1) and class IIb (HDAC6) inhibitory potency in the living cell environment17. Another advantage of the method is that it can easily be adapted to different cell lines, such as neuroblasts23, with the possibility for miniaturization and automation, as previously described for other cell-based assays24. The detection of specific substrates by mass spectrometry contributes to the accurate measurement of the deacetylated products, providing comparable quantitative results. This is the first method available for determining IC50 values towards specific HDAC isoforms in living cells. As a matter of comparison, the non-quantitative selective profile of HDAC inhibitors observed by western blot have been demonstrated to be in line with the selectivity profile obtained with the method described here17. Even if this method has been developed for the discovery of new class-selective HDAC inhibitors, it could also be applied to measure HDAC activity within cell models over the course of a disease. This could contribute to the elucidation of epigenetic and molecular mechanisms in relevant diseases and could potentially identify drug candidates. In view of extrapolating this method towards HDAC isoforms other than HDAC1 and HDAC6, the development of new selective HDAC substrates is strongly encouraged. Moreover, the substrate specificity of MOCPAC and BATCP should be fully characterized in terms of targeting single isoforms. Indeed, while MOCPAC is classified as an HDAC1-specific substrate (class I over class II), it is more likely that it is a generic class I substrate (including HDAC3 isoform), as previously demonstrated25. Accordingly, BATCP is a well-known HDAC6-selective substrate (class II over class I HDACs), but its specificity towards other class II HDACs, such as HDAC4, deserves further characterization25.
Enzymatic activity may differ depending upon the cell line, cell number, confluency, and incubation time. When a cell line is used for the first time in the assay, it is important to test a range of substrate concentrations in control cells, at different time points, to analyze the enzyme kinetics by UHPLC-MS (e.g., through a Michaelis-Menten plot). This will allow researchers to choose the most suitable incubation time and substrate concentration. This choice relies on two major parameters: 1) the enzyme kinetics and 2) the detection of the substrates and their deacetylated products. When analyzing enzyme kinetics, it is essential to evaluate the initial velocity values (i.e., when the product formation is within the linear range) to establish the enzyme kinetics. According to the kinetics, substrate concentrations under the Km should be chosen to provide an accurate percentage of inhibition, especially in the case of competitive and uncompetitive inhibitors26. On the other hand, the chosen substrate concentration should provide detectable and quantifiable MS signals for both the substrate and the deacetylated product (signal-to-noise ratio &#8805;20)17. When using multiple substrates, the chromatographic method should ensure the separation of all peaks to be analyzed. Alternative chromatographic systems may require changes in the separation gradient and column type.

HDACs belong to a family of enzymes able to deacetylate histones within the chromatin structure. They also have other protein substrates in the cytosol and are located in various cell compartments. A total of 18 HDAC isoforms have been identified so far and have been related to several cell mechanisms, including the regulation of transcription factors and gene expression, as well as cell signaling and transport1,2,3,4,5,6,7. HDAC catalytic inhibitors have emerged as potential therapeutic drugs for cancer therapy. Most HDAC inhibitors currently approved by the FDA are for the treatment of T-cell lymphoma and multiple myeloma, and are non-specific HDAC inhibitors, such as vorinostat (SAHA), belinostat, and panobinostat8,9. However, a series of side effects have been associated with pan-inhibitors, and the search for isoform-specific small molecules is a hot topic in medicinal chemistry and drug discovery. Accordingly, the class I (HDAC1-3 and HDAC8) selective inhibitor romidepsin is an already approved drug10, while HDAC6-specific inhibitors are currently under clinical trials, with increased therapeutic potential in multiple myeloma11,12,13,14,15.
Screening assays to characterize HDAC inhibitors are based on the incubation of an HDAC substrate with an enzymatic source (single isoform, nuclear extract, or cell lysate). The substrate is usually a small peptide sequence containing an acetyl lysine residue coupled to a cleavable fluorophore (e.g., coumarin), such as N-(4-methyl-7-aminocoumarinyl)-N&#945;-(t-butoxycarbonyl)-N&#969;-acetyllysineamide (MAL)16. To distinguish between isoform-specific activities, separate cell-free assays involving each isoform are necessary and might not reflect the real isoform activity in living cells. Isoform-specific substrates are commercially available, such as benzyl (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC, HDAC1 specific substrate) and (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic acid tert-butyl ester (BATCP, HDAC6 specific substrate) (Figure 1B). However, a multi-substrate mixture containing MAL, MOCPAC, and BATCP given to living cells will not permit the detection of the individual deacetylated products by fluorometric measurement, given that they bear the same cleavable fluorophore.
The method described here allows for the detection and relative quantification of each substrate and its deacetylated product in HeLa cells using a multi-substrate assay followed by UHPLC-ESI-MS/MS analysis17. An HDAC assay is conducted on HeLa cells to enable the direct identification of HDAC inhibitory activity and of the specificity of test compounds on endogenous HDACs. There is a focus on HDAC1 and HDAC6, which are simultaneously evaluated. To achieve these enzymatic measurements in a single incubation assay, a mixture of non-specific and specific HDAC substrates is added to treated and untreated HeLa cells plated on a 96-well plate. Following an incubation step, cells are lysed to release the substrates and their respective reaction products, which are separated and detected using a UHPLC-MS method (Figure 1C). The deacetylated products of the MAL, MOCPAC, and BATCP substrates are the deacetylated MAL (dMAL), deacetylated MOCPAC (dMOCPAC), and deacetylated BATCP (dBATCP), respectively. Dose-response curves can be built with active compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg" /> 
Figure 1: General scheme for the cell-based HDAC assay to identify HDAC1- and HDAC6-specific inhibitors by the UHPLC-MS analysis of multiple substrates. (A) Scheme of a typical 96-well plate containing treated (test compounds) and untreated (control) HeLa cells, as well as cell-free blanks. (B) Chemical structure of substrates added as a mixture (21 &#181;M each) to be deacetylated by endogenous HDACs. (C) Typical UHPLC-MS chromatogram showing the peaks of the added substrates (MAL, MOCPAC, and BATCP) and their deacetylated products (dMAL, dMOCPAC, and dBACTP, respectively). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>BATCP</td><td>Sigma-Aldrich</td><td>B4061</td><td></td></tr><tr><td>MAL</td><td>Sigma-Aldrich</td><td>SCP0168</td><td>Synonym: BOC-Ac-Lys-AMC</td></tr><tr><td>MOCPAC</td><td>Sigma-Aldrich</td><td>M2195</td><td></td></tr><tr><td>MS275</td><td>Sigma-Aldrich</td><td>EPS002</td><td></td></tr><tr><td>Trichostatin A</td><td>Sigma-Aldrich</td><td>T8552</td><td></td></tr><tr><td>Tubastatin A</td><td>Sigma-Aldrich</td><td>SML0044</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific</td><td>10660131</td><td></td></tr><tr><td>Formic acid, LC/MS grade</td><td>Fisher Scientific</td><td>10596814</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, HPLC grade</td><td></td><td></td><td>distilled H&lt;sub&gt;2&lt;/sub&gt;O filtered through a Milli-Q purification system</td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>ATCC CRM-CCL-2</td><td></td></tr><tr><td>Cell dissociation reagent TrypLE Express</td><td>ThermoFisher Scientific</td><td>12604013</td><td></td></tr><tr><td>DMSO, cell culture grade</td><td>Applichem</td><td>146463</td><td></td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Biowest</td><td>S1810</td><td></td></tr><tr><td>MEM</td><td>ThermoFisher Scientific</td><td>22561021</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Bioconcept</td><td>4-01F00-H</td><td></td></tr><tr><td>10x RIPA buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>SigmaFast protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>S8820</td><td></td></tr><tr><td>96-well plates, sterile, flat-bottom, tissue culture treated Corning</td><td>VWR</td><td>29442-058</td><td></td></tr><tr><td>96-well plates, non-sterile, V-bottom Corning</td><td>VWR</td><td>29442-404</td><td>used in the centrifugation step</td></tr><tr><td>96-well plate, conical bottom, Nunc</td><td>ThermoFisher Scientific</td><td>249944</td><td>compatible with the Acquity UHPLC system</td></tr><tr><td>T75 cell culture flasks Corning</td><td>Sigma-Aldrich</td><td>CLS430641</td><td></td></tr><tr><td>Peelable heat sealing foil</td><td>Waters</td><td>186002789</td><td></td></tr><tr><td>Acquity UPLC system</td><td>Waters</td><td></td><td></td></tr><tr><td>Eppendorf Centrifuge 5810 R</td><td>Fisher Scientific</td><td>05-413-323</td><td></td></tr><tr><td>Integration software: MassLynx V4.1</td><td>Waters</td><td></td><td>Catalog number not available</td></tr><tr><td>Combi thermo-sealer SP-0669/240</td><td>Waters</td><td></td><td>Catalog number not available</td></tr><tr><td>Quattro micro API Tandem Quadrupole System</td><td>Waters</td><td></td><td>Catalog number not available</td></tr></tbody></table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

To demonstrate the application of the method to identifying non-selective and selective inhibitors for HDAC1 (class I) and HDAC6 (class IIb), HeLa cells were treated with known standard compounds: trichostatin A (non-selective between HDAC1 and HDAC6)18, MS275 (HDAC1-selective inhibitor)19, and tubastatin A (HDAC6-selective inhibitor)20. By using the present method (Figure 1), IC50 values could be directly determined in living cells for HDAC1 (by looking at MOCPAC deacetylation) and for HDAC6 (by looking at BATCP deacetylation) (Figure 2). Trichostatin A was a potent but non-selective inhibitor towards HDAC1 and HDAC6. On the other hand, MS275 was selective for HDAC1, while tubastatin A was clearly selective for HDAC6.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55878/55878fig2.jpg" /> 
Figure 2: IC50 Dose-response curves simultaneously obtained for HDAC1 and HDAC6 of three standard HDAC inhibitors: the non-selective trichostatin A, the HDAC1-selective MS275, and the HDAC6-selective tubastatin A. IC50 values presented in the adjacent table are the mean &#177; SD of three independent measurements. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55878/55878fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Injection volume</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td>Column</td>
			<td>C18 (2.6 &#956;m, 100 mm x 3 mm i.d.)</td>
		</tr>
		<tr>
			<td>Column temperature</td>
			<td>40 &#176;C</td>
		</tr>
		<tr>
			<td>Elution flow rate</td>
			<td>0.8 mL/min (typical pressure range: 400-600 bars)</td>
		</tr>
		<tr>
			<td>Elution gradient</td>
			<td>5-45% B in 10 min</td>
		</tr>
		<tr>
			<td></td>
			<td>45-98% B in 2 min</td>
		</tr>
		<tr>
			<td>Wash step</td>
			<td>98% B for 2 min</td>
		</tr>
		<tr>
			<td>Equilibrating step</td>
			<td>5% B for 4 min</td>
		</tr>
		<tr>
			<td>ESI-MS/MS</td>
			<td>cone voltage, 30 V; capillary source voltage, 3.0 kV; capillary temperature, 350 &#176;C; source temperature, 120 &#176;C; sheath gas - nitrogen (600 L/h); collision gas pressure, argon set to 3 x 10-3 mbar</td>
		</tr>
		<tr>
			<td></td>
			<td>Collision parameters and reaction channels optimized with a dwell time of 0.1 s, 0.01 s of interscan delays and collision energy set at 20 eV.</td>
		</tr>
		<tr>
			<td></td>
			<td>MAL detection with mass transitions 446 &#8594; 346 (MAL) and 404 &#8594; 304 (dMAL).&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>MOCPAC detection with mass transitions 494 &#8594; 449 (MOCPAC) and 439 &#8594; 394 (dMOCPAC).&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td>BATCP detection with mass transitions 500 &#8594; 400 (BATCP) and 476 &#8594; 376 (dBATCP).</td>
		</tr>
	</tbody>
</table>
Table 1: UHPLC-ESI-MS/MS conditions.

Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

simultaneous-measurement-hdac1-hdac6-activity-hela-cells-using-uhplc

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.5K Views.  Universités des Geneva–Lausanne (EPGL). De huidige methode dient ter identificatie van isovorm-specifieke remmers van Histon deacetylases (HDAC) in HeLa cellen door de UHPLC-MS-analyse van meerdere substraten. Dit is een antilichaam-vrije methode ontwikkeld om te reflecteren van HDAC1 en HDAC6 activiteit in de levende cel omgeving, in tegenstelling tot één-isovorm cel-gratis testen.

Video: Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Biochemistry

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Cancer Research

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Biology

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video