Name: analysis of histone antibody specificity with peptide microarrays
Uploaded: 2017-08-01T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

This work was supported in part by the Van Andel Research Institute and a research grant from the National Institutes of Health (CA181343) to S.B.R.

1. Installing and Running ArrayNinja
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Antibody reliability in biomedical research applications is paramount46,47. This is especially true in chromatin biochemistry given the position of antibodies as key tools for the majority of techniques developed to characterize the abundance and distribution of histone PTMs. The protocol presented here details an optimized pipeline for the design, fabrication, and use of peptide microarrays to analyze histone PTM antibody specificity. This pipeline has been used...

Genomic DNA is elegantly packaged inside the eukaryotic cell nucleus with histone proteins to form chromatin. The repeating subunit of chromatin is the nucleosome, which consists of 147 base pairs of DNA wrapped around an octameric core of histone proteins &#8211; H2A, H2B, H3, and H41. Chromatin is broadly organized into loosely packed euchromatin and tightly packed heterochromatin domains. The degree of chromatin compaction regulates the extent to which protein machineries can access the underlying DNA to carry out fundamental DNA-templated processes like replication, transcription, and repair.
Key regulators of genome accessibility in the context of chromatin are PTMs on the unstructured tail and core domains of histone proteins2,3. Histone PTMs function directly by influencing the structure of chromatin4 and indirectly through the recruitment of reader proteins and their associated macromolecular complexes that have chromatin remodeling, enzymatic, and scaffolding activities5. Studies of histone PTM function over the past two decades overwhelmingly suggest these marks play key roles in regulating cell fate, organismal development, and disease initiation/progression. Fueled by advances in mass spectrometry-based proteomic technology, more than 20 unique histone PTMs on more than 80 distinct histone residues have been discovered6. Notably, these modifications often occur in combinations, and consistent with the &#34;histone code&#34; hypothesis, numerous studies suggest that reader proteins are targeted to discrete regions of chromatin through recognition of specific combinations of histone PTMs7,8,9. A key challenge moving forward will be to assign functions to the growing list of histone PTMs and to determine how specific combinations of histone PTMs orchestrate the dynamic functions associated with chromatin.
Antibodies are the lynchpin reagents for the detection of histone PTMs. As such, more than 1,000 histone PTM-specific antibodies have been commercially developed for use in chromatin biochemistry research. With the rapid development of high-throughput DNA sequencing technology, these reagents are being used extensively by individual investigators and large-scale epigenomics &#34;roadmap&#34; initiatives (e.g., ENCODE and BLUEPRINT) in ChIP-seq (chromatin immunoprecipitation coupled with next-generation sequencing) pipelines to generate high-resolution spatial maps of histone PTM distribution genome-wide10,11. However, recent studies have shown that the specificity of histone PTM antibodies can be highly variable and that these reagents exhibit unfavorable properties such as off-target epitope recognition, strong positive and negative influence by neighboring PTMs, and difficulty discriminating the modification order on a particular residue (e.g., mono-, di-, or tri-methyllysine)12,13,14,15,16,17,18. Therefore, rigorous quality control of histone PTM-specific antibody reagents is necessary to accurately interpret data generated with these valuable reagents.
Microarray technology enables the simultaneous interrogation of thousands of macromolecular interactions in a high-throughput, reproducible, and miniaturized format. For this reason, a variety of microarray platforms have been created to analyze protein-DNA19,20, protein-protein 21, and protein-peptide interactions22. Indeed, histone peptide microarrays have emerged as an informative discovery platform for chromatin biochemistry research, enabling high-throughput profiling of the writers, erasers, and readers of histone PTMs15,23,24, and also for the analysis of histone antibody specificity17,25. Beyond their application in chromatin and epigenetics research, histone peptide arrays have potential utility as a diagnostic/prognostic test for systemic lupus erythematosus and other autoimmune diseases where anti-chromatin autoantibodies are generated26,27.
Here, we describe an integrated pipeline that we have developed for designing, fabricating, and querying histone peptide microarrays to generate specificity profiles for antibodies that recognize histones and their PTMs. The pipeline is facilitated by ArrayNinja, an open-source, interactive software application that we recently developed, which integrates the design and analysis stages of microarray experiments28. ArrayNinja works best in Google Chrome. Briefly, a robotic contact microarray printer is used to deposit a library of biotin-conjugated histone peptides at defined positions on streptavidin-coated glass microscope slides. Arrays can then be used in a competitive and parallel assay format to interrogate antibody-epitope interactions (Figure 1). The peptide library consists of hundreds of unique synthetic peptides harboring PTMs (lysine acetylation, lysine/arginine methylation, and serine/threonine phosphorylation) alone and in relevant combinations largely derived from proteomics datasets. Methods for peptide synthesis and validation are detailed elsewhere23. Data generated from our ongoing histone PTM antibody screening efforts utilizing this array platform are archived on a public web resource, the Histone Antibody Specificity Database (www.histoneantibodies.com). Notably, histone peptide microarrays fabricated with variations of this protocol have also been used extensively to characterize the activity of histone PTM reader domains8,29,30,31,32,33,34,35,36,37 and more recently to profile histone PTM writer and eraser activities24.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55912/55912fig1.jpg" /> 
Figure 1: Cartoon Depiction of the Stepwise Procedure for Antibody Screening on a Histone Peptide microarray. Biotinylated histone peptides harboring defined post-translational modifications (red and blue circles) are co-printed with biotin-fluorescein on streptavidin-coated glass. Positive interactions are visualized as red fluorescence. <a href="http://cloudflare.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="746">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Printing Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">PPB</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BSA</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">2390</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Streptavidin-coated glass microscope slides</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td>439003-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">polypropylene 384 well plate</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td align="right" style="width:105px;">784201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Biotin-fluorescein</td>
			<td style="width:107px;">Sigma</td>
			<td align="right" style="width:105px;">53608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Aushon</td>
			<td align="right" style="width:105px;">2470</td>
			<td style="width:281px;">Aushon 2470 Microarray Printer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Gene Machines</td>
			<td style="width:105px;">OmniGrid 100</td>
			<td style="width:281px;">OmniGrid Microarray Printer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:107px;">Invitrogen</td>
			<td align="right" style="width:105px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Blocking Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">SBB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Hydrophobic wax pen</td>
			<td style="width:107px;">Vector Labs</td>
			<td style="width:105px;">H-4000</td>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silicon Gasket</td>
			<td style="width:107px;">Grace Bio-labs</td>
			<td align="right" style="width:105px;">622511</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Hybridization Vessel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td align="right">267061</td>
			<td>or similar vessel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21244</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-rabbit)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21235</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax Imprinter</td>
			<td style="width:107px;">ArrayIt</td>
			<td>MSI48</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween-20</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">9490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microarray Scanner</td>
			<td style="width:107px;">Innopsys</td>
			<td style="width:105px;">InnoScan 1100AL</td>
			<td>or equivalent microarray scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EipTitan Histone Peptide Microarray</td>
			<td style="width:107px;">Epicypher</td>
			<td align="right" style="width:105px;">112001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">AbSurance Pro Histone Peptide Microarray</td>
			<td style="width:107px;">Millipore</td>
			<td align="right" style="width:105px;">16668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">MODified Histone Peptide Array</td>
			<td style="width:107px;">Active Motif</td>
			<td align="right" style="width:105px;">13001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Histone Code Peptide Microarrays</td>
			<td style="width:107px;">JPT</td>
			<td style="width:105px;">His_MA_01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax</td>
			<td style="width:107px;">Royal Oak</td>
			<td style="width:105px;">GulfWax</td>
			<td>for wax imprinter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Humidified Microarray Slide Hybridization Chamber</td>
			<td style="width:107px;">VWR</td>
			<td style="width:105px;">97000-284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">High throughput microscope slide washing chamber</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">HTW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microscope slide centrifuge</td>
			<td style="width:107px;">VWR</td>
			<td>93000-204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 1</td>
			<td>Abcam</td>
			<td align="right" style="width:105px;">8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 2</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:105px;">07-473</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:255px;">Biotinylated histone peptide</td>
			<td style="width:107px;">EpiCypher</td>
			<td>12-0001</td>
			<td style="width:281px;">Example peptide. Similar peptides with various modifications are available from several commercial sources.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ImageMagick</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://www.imagemagick.org/script/index.php</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ArrayNinja</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://rothbartlab.vai.org/tools/</td>
		</tr>
	</tbody>
</table>

analysis of histone antibody specificity with peptide microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This protocol has been used to design and fabricate a peptide microarray platform for the analysis of histone PTM antibody specificity. The array queries a library of more than 300 unique peptide features (20 - 40 residues in length) representing many of the known combinations of PTMs found on core and variant histone proteins38. This pipeline has been a workhorse for the screening of many widely used and commercially available histone PTM antibodies, and full data...

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

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