This work was supported by funding from NIH grants (DP2OD007447, R01GM110174 and R01AI118891).

<ol>
	<li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canalization+of+development+and+the+inheritance+of+acquired+characters.">Canalization of development and the inheritance of acquired characters.</a> Nature. 150, 563-565 (1942).</li><li>Sharma, S., Kelly, T. K., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+cancer.">Epigenetics in cancer.</a> Carcinogenesis. 31 (1), 27-36 (2010).</li><li>Reik, W., Dean, W., Walter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+in+mammalian+development.">Epigenetic reprogramming in mammalian development.</a> Science. 293 (5532), 1089-1093 (2001).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128 (4), 693-705 (2007).</li><li>Tessarz, P., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+core+modifications+regulating+nucleosome+structure+and+dynamics.">Histone core modifications regulating nucleosome structure and dynamics.</a> Nat Rev Mol Cell Bio. 15 (11), 703-708 (2014).</li><li>Fischle, W., Wang, Y. M., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+and+chromatin+cross-talk.">Histone and chromatin cross-talk.</a> Curr Opin Cell Biol. 15 (2), 172-183 (2003).</li><li>Lee, J. S., Smith, E., Shilatifard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+language+of+histone+crosstalk.">The language of histone crosstalk.</a> Cell. 142 (5), 682-685 (2010).</li><li>Simboeck, E., 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=A+Phosphorylation+Switch+Regulates+the+Transcriptional+Activation+of+Cell+Cycle+Regulator+p21+by+Histone+Deacetylase+Inhibitors.">A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors.</a> J Biol Chem. 285 (52), 41062-41073 (2010).</li><li>Hirota, T., Lipp, J. J., Toh, B. H., Peters, 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=Histone+H3+serine+10+phosphorylation+by+Aurora+B+causes+HP1+dissociation+from+heterochromatin.">Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.</a> Nature. 438 (7071), 1176-1180 (2005).</li><li>Xhemalce, B., Kouzarides, 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+chromodomain+switch+mediated+by+histone+H3+Lys+4+acetylation+regulates+heterochromatin+assembly.">A chromodomain switch mediated by histone H3 Lys 4 acetylation regulates heterochromatin assembly.</a> Genes Dev. 24 (7), 647-652 (2010).</li><li>Vermeulen, 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=Quantitative+interaction+proteomics+and+genome-wide+profiling+of+epigenetic+histone+marks+and+their+readers.">Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.</a> Cell. 142 (6), 967-980 (2010).</li><li>van Attikum, H., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+histone+modifications+during+the+DNA+damage+response.">Crosstalk between histone modifications during the DNA damage response.</a> Trends Cell Biol. 19 (5), 207-217 (2009).</li><li>Fernandez-Capetillo, O., 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=H2AX+is+required+for+chromatin+remodeling+and+inactivation+of+sex+chromosomes+in+male+mouse+meiosis.">H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis.</a> Dev Cell. 4 (4), 497-508 (2003).</li><li>Santaguida, S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+and+miracles+of+kinetochores.">The life and miracles of kinetochores.</a> Embo J. 28 (17), 2511-2531 (2009).</li><li>Egelhofer, T. 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=An+assessment+of+histone-modification+antibody+quality.">An assessment of histone-modification antibody quality.</a> Nat Struct Mol Biol. 18 (1), 91-93 (2011).</li><li>Sidoli, S., Cheng, L., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+in+chromatin+biology+and+epigenetics:+Elucidation+of+post-translational+modifications+of+histone+proteins+by+mass+spectrometry.">Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.</a> J Proteomics. 75 (12), 3419-3433 (2012).</li><li>Plazas-Mayorca, M. D., 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=One-Pot+Shotgun+Quantitative+Mass+Spectrometry+Characterization+of+Histones.">One-Pot Shotgun Quantitative Mass Spectrometry Characterization of Histones.</a> J Proteome Res. 8 (11), 5367-5374 (2009).</li><li>Sidoli, S., 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=Drawbacks+in+the+use+of+unconventional+hydrophobic+anhydrides+for+histone+derivatization+in+bottom-up+proteomics+PTM+analysis.">Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis.</a> Proteomics. 15 (9), 1459-1469 (2015).</li><li>Lin, S., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+histone+posttranslational+modification+patterns+by+high-resolution+mass+spectrometry.">Examining histone posttranslational modification patterns by high-resolution mass spectrometry.</a> Methods Enzymol. 512, 3-28 (2012).</li><li>Sidoli, S., 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=SWATH+Analysis+for+Characterization+and+Quantification+of+Histone+Post-translational+Modifications.">SWATH Analysis for Characterization and Quantification of Histone Post-translational Modifications.</a> Mol Cell Proteomics. , (2015).</li><li>Krautkramer, K. A., Reiter, L., Denu, J. M., Dowell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+SAHA-Dependent+Changes+in+Histone+Modifications+Using+Data-Independent+Acquisition+Mass+Spectrometry.">Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry.</a> J Proteome Res. , (2015).</li><li>Yuan, Z. F., 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=EpiProfile+Quantifies+Histone+Peptides+With+Modifications+by+Extracting+Retention+Time+and+Intensity+in+High-resolution+Mass+Spectra.">EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra.</a> Mol Cell Proteomics. 14 (6), 1696-1707 (2015).</li><li>MacLean, 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=Skyline:+an+open+source+document+editor+for+creating+and+analyzing+targeted+proteomics+experiments.">Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.</a> Bioinformatics. 26 (7), 966-968 (2010).</li><li>Yuan, Z. F., Lin, S., Molden, R. C., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+proteomic+search+engines+for+the+analysis+of+histone+modifications.">Evaluation of proteomic search engines for the analysis of histone modifications.</a> J Proteome Res. 13 (10), 4470-4478 (2014).</li><li>Tan, Y., Xue, Y., Song, C., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylated+histone+H3K56+interacts+with+Oct4+to+promote+mouse+embryonic+stem+cell+pluripotency.">Acetylated histone H3K56 interacts with Oct4 to promote mouse embryonic stem cell pluripotency.</a> Proc Natl Acad Sci U S A. 110 (28), 11493-11498 (2013).</li><li>Vonholt, 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=Isolation+and+Characterization+of+Histones.">Isolation and Characterization of Histones.</a> Methods Enzymol. 170, 431-523 (1989).</li><li>Zhang, K. L., 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=Identification+of+acetylation+and+methylation+sites+of+histone+H3+from+chicken+erythrocytes+by+high-accuracy+matrix-assisted+laser+desorption+ionization-time-of-flight,+matrix-assisted+laser+desorption+ionization-postsource+decay,+and+nanoelectrospray+ionization+tandem+mass+spectrometry.">Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry.</a> Anal. Biochem. 306 (2), 259-269 (2002).</li><li>Jufvas, A., Stralfors, P., Vener, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+Variants+and+Their+Post-Translational+Modifications+in+Primary+Human+Fat+Cells.">Histone Variants and Their Post-Translational Modifications in Primary Human Fat Cells.</a> Plos One. 6 (1), e15960 (2011).</li><li>Bonaldi, T., Imhof, A., Regula, J. 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+combination+of+different+mass+spectroscopic+techniques+for+the+analysis+of+dynamic+changes+of+histone+modifications.">A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications.</a> Proteomics. 4 (5), 1382-1396 (2004).</li><li>Zhao, X. L., 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=Comparative+Proteomic+Analysis+of+Histone+Post-translational+Modifications+upon+Ischemia/Reperfusion-Induced+Retinal+Injury.">Comparative Proteomic Analysis of Histone Post-translational Modifications upon Ischemia/Reperfusion-Induced Retinal Injury.</a> J Proteome Res. 13 (4), 2175-2186 (2014).</li><li>Lin, S., 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=Stable-isotope-labeled+histone+peptide+library+for+histone+post-translational+modification+and+variant+quantification+by+mass+spectrometry.">Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry.</a> Mol Cell Proteomics. 13 (9), 2450-2466 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The protocol described here is optimized considering costs, time, and performance. Other preparations are possible, but they have limitations, especially in the case of coupling with MS analysis. For instance, the high-salt extraction protocol can be used to purify histones26 instead of TCA precipitation (section 3). High-salt protocol is intrinsically milder, as it does not use strong acid. This preserves acid-labile PTMs and increases the yield of extracted histones, as TCA precipitation co-precipitates many other chromatin binding proteins. However, high-salt extraction leads to samples containing too concentrated salt for HPLC-MS/MS. In an alternative preparation, histone digestion can be performed without propionylation (section 6 - 8), for instance by reducing trypsin incubation time and the enzyme/substrate ratio27 or using ArgC as digestion enzyme28-30. However, derivatization with propionic anhydride is recommended, as it leads to the generation of more hydrophobic peptides, which are better retained during liquid chromatography.
For chemical derivatization, a variety of organic acid anhydrides have been evaluated and their merits comprehensively discussed18. Nonetheless, propionic anhydride proved to the best compromise between efficiency, minimized side products and improved peptide hydrophobicity. Potentially, propionic anhydride can be purchased in the isotopically labeled form; this allows for multiplexing analysis due to the possibility of mixing multiple samples and discriminate them at the MS level based on the different masses imparted from the heavy label. However, this analysis leads to an increased complexity of the LC-MS chromatogram and reduces the amount of sample that can be injected for each single condition.
In this regard, some critical aspects of the protocol should be highlighted. The following should be used as checklist to find errors in performing the procedure in case negative results are obtained. First, after nuclei precipitation the pellet should be carefully washed with NIB without NP-40 Alternative (section 2.10) until complete removal of the detergent (noticeable by the lack of bubbles during mixing). Failing to do so would compromise histone extraction with acids. Second, after histone precipitation with TCA (section 3.9) washes of the pellet with acetone is crucial. Presence of concentrated acid would harm the following step if propionylation and digestion (section 6.1) are directly performed. It would be not problematic in case histone fractionation is performed (section 5). Third, it is essential that the propionylation reaction is performed quickly (section 6.3 - 6.7). To do so, avoid using the same propionylation mix (propionic anhydride + acetonitrile) for more than 3 - 4 consecutive samples. Additionally, pH is the most important aspect of trypsin digestion (section 7). If not around 8.0 (7.5 - 8.5) the digestion will be ineffective. This can happen, as the sample will be rich in propionic acid at this step. NH4OH can be added until necessary. Also, for researchers familiar with proteomics workflows it will feel normal to acidify the sample to terminate trypsin digestion. This should not be done, as it will jeopardize the following reaction, i.e., propionylation of peptide N-termini (section 8.1). Finally, in the same issue, it is important to remember for data analysis that unmodified peptides are not actually unmodified; all free lysine residues and N-termini will be occupied by propionylation (56.026 Da). Thus, performing extracting ion chromatography of the mass corresponding uniquely to the peptide sequence would lead to no results.
The limitations of the method are mostly related to the inability of detecting combinatorial PTMs, due to the short peptide sequences, and the biases in achieving the true abundance of a modification, due to the fact that peptides in different modified forms might ionize with different efficiencies. The first issue can be solved by combining this technique with a middle-down or top-down approach (reviewed in 16). This type of analysis, even if technically more challenging, is ideal for studying co-existence frequencies of modifications. Moreover, it allows better discrimination of histone variants, which cannot always be achieved with bottom-up since some peptides have the same sequence in different histone variants. The second issue, related to the ionization efficiency, can be solved using a library of synthetic peptides31. This approach ensures a more accurate estimation of the relative abundance of histone PTMs. However, in most experiments, the desired outcome is the relative changes of given modifications between analyzed conditions. In this case, such correction is not necessary, due to the fact that all samples have the same bias.
In conclusion, this protocol allows for the analysis of histone PTMs that can be completed in 3 days using nLC coupled to tandem MS. Comparisons with techniques other than MS, i.e., using antibody based strategies as discussed in the Introduction, are not suitable, as they cannot achieve even nearly this level of throughput. In addition, antibody based techniques do not allow for the discovery of novel modifications, but they are exclusively based on confirming and quantifying predicted marks. We thus speculate that bottom-up proteomics on histone peptides will gain popularity in proteomics laboratories due to the intuitive advantages in knowing the regulation of histone marks, which are protagonists in tuning gene expression and thus affect the regulation of the proteome. Moreover, the protocol described includes recent improvements in the sample preparation and software for data analysis, which make histone analysis more trivial also for laboratories that never experienced characterization of this type of hypermodified peptides.

Epigenetics is defined as the study of heritable changes in gene expression that arise by mechanisms other than altering the underlying DNA sequence1. Epigenetic regulation is critical during development as the organism undergoes dramatic phenotypic changes even though its DNA content does not change. There are several critical components required for proper epigenetic maintenance, including histone post-translational modifications (PTMs), histone variants, non-coding RNAs, DNA methylation and DNA binding factors, each of which affect gene expression through different mechanisms2. For example, while DNA methylation is a highly stable modification that represses gene translation3, histone variants and histone PTMs are much more dynamic and can influence chromatin in a variety of ways4.
Histone PTMs are mostly localized on the N-terminal tails, as they are the most exposed and flexible region of the protein. However, the nucleosome core is also heavily modified compared to average proteins5. Even though histone marks have been extensively characterized in the last decade, many links between known histone marks and their function are still unclear. This is largely due to the fact that most histone PTMs do not work alone, but rather function in tandem with other PTMs (&#34;cross-talk&#34;) to alter a specific process such as transcription6,7. For instance, the combinatorial mark H3S10K14ac on the gene p21 activates its transcription, which would not occur with only one of the two PTMs8. The protein HP1 compacts chromatin by recognizing H3K9me2/me3 and spreading the modification to nearby nucleosomes. However, HP1 cannot bind H3K9me2/3 when the adjacent S10 is phosphorylated9. Acetylation of H3K4 inhibits binding of the protein spChp1 to H3K9me2/me3 in Schizosaccharomyces pombe10. Furthermore, the histone lysine demethylase PHF8 has the highest nucleosome binding efficiency when three PTMs H3K4me3, K9ac, and K14ac are present11. These examples highlight the importance of achieving a global overview of histone PTM changes rather than focusing on single modifications.
The presence of sequence variants also increases the complexity of histone analysis, as histone isotypes generally have highly similar sequences, but often have different roles in chromatin. For example, H2A.x has a C-terminal sequence which is more easily phosphorylated upon DNA damage compared to canonical H2A12, and it is required for inactivation of sex chromosomes in male mouse meiosis13; similarly, CENP-A substitutes canonical histone H3 in centromeres14. Despite their different functions, these variants share a large portion of their amino acid sequence with the respective canonical histone, making it difficult to identify and quantify them separately.
Antibody-based techniques such as western blotting have been extensively adopted to characterize histones. However, antibody-based approaches are limited for the following reasons: (i) they can only confirm the presence of a modification and cannot identify unknown PTMs; (ii) they are biased due to the presence of co-existing marks, which can influence binding affinity; (iii) they cannot identify combinatorial marks, as only very few antibodies are available for such purpose and (iv) they cross-react between highly similar histone variants or similar PTMs (e.g., di- and trimethylation of lysine residues). Egelhofer et al. described that more than 25% of commercial antibodies fail specificity tests by dot blot or western blot, and among specific antibodies more than 20% fail in chromatin immunoprecipitation experiments15. Mass spectrometry (MS) is currently the most suitable analytical tool to study novel and/or combinatorial PTMs, and it has been extensively implemented for histone proteins (reviewed in 16). This is mostly due to high sensitivity and mass accuracy of MS, and the possibility to perform large-scale analyses.
The bottom-up strategy is the most commonly used MS-based proteomics strategy for histone characterization and their PTMs, wherein the intact protein is enzymatically digested into short peptides (5 - 20 aa). This digestion facilitates both LC separation and MS detection. Masses in the range of 600 - 2,000 Da are commonly more easily ionized and identified with higher mass accuracy and resolution than larger masses. MS/MS fragmentation is also improved, as short peptides are generally well-suited for collision induced dissociation (CID). However, histones present a challenge for bottom-up MS as they are highly enriched in basic amino acid residues, namely lysine and arginine. Therefore, trypsin digestion leads to the generation of peptides that are too small for LC retention and unambiguous localization of the PTMs. To circumvent this issue, our protocol includes lysine and peptide N-terminal chemical derivatization17. The use of propionic anhydride is recommended for efficient chemical derivatization as compared to other reagents 18. Such derivatization blocks the &#603;-amino groups of unmodified and monomethyl lysine residues, allowing trypsin to perform proteolysis only at the C-terminal of arginine residues. Derivatized amines cannot exchange protons with the solution and thus the peptides are generally only doubly or triply charged, facilitating MS and MS/MS detection. Moreover, N-terminal derivatization increases peptide hydrophobicity and thus reversed-phase chromatographic retention. Here, we describe the workflow to purify histones and prepare them for PTM analysis via bottom-up proteomics (Figure 1). This strategy achieves quantification of single histone marks and combinatorial marks for histone PTMs that are relatively close in the amino acid sequence.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Trypsin 0.25% EDTA</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">25200056</td>
			<td style="width:217px;">For harvesting cells</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">PBS</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">14200075</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Tris</td>
			<td style="width:157px;">Roche</td>
			<td style="width:151px;">77-86-1</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Potassium Chloride</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sodium Chloride</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">S9888</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Magnesium Chloride hexahydrate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">M9272</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Calcium Chloride, anhydrous</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">C1016</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sucrose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">BP220-1</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">DTT</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">15508-013</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">AEBSF</td>
			<td style="width:157px;">EMD Millipore Corp</td>
			<td style="width:151px;">101500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Microcystin</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">M4194</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sodium Butyrate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">B5887</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X)&#160;</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">78445</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">NP-40 Alternative</td>
			<td style="width:157px;">CALBIOCHEM</td>
			<td style="width:151px;">492016</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sulfuric Acid, ACS grade</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">7664-93-9</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Trichloroacetic acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">T6399</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Acetone</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">179124</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HCl</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">A144-500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Bradford reagent</td>
			<td style="width:157px;">Biorad</td>
			<td style="width:151px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">30% acrylamide/bis 29:1 -- 500ml</td>
			<td style="width:157px;">Biorad</td>
			<td style="width:151px;">1610156</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Coomassie</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">20278</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 Column (5um) 2.1mm x 250mm</td>
			<td style="width:157px;">Grace</td>
			<td style="width:151px;">218TP52</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 Column (5um) 4.6mm x 250mm</td>
			<td style="width:157px;">Grace</td>
			<td style="width:151px;">218TP54</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HPLC grade acetonitrile</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">A955-4</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HPLC grade water</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">W6 4</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">TFA</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">A11650</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Ammonium Bicarbonate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">A6141</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">ammonium hydroxide</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">338818</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">propionic anhydride</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">240311</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sequencing grade modified trypsin</td>
			<td style="width:157px;">Promega</td>
			<td style="width:151px;">PRV5113</td>
			<td>For digesting histones for MS</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Acetic Acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">49199</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 extraction disk</td>
			<td style="width:157px;">Empore</td>
			<td style="width:151px;">2215</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Formic Acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">F0507</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

As an example, we analyzed histones extracted from human embryonic stem cells (hESCs) with and without retinoic acid (RA) stimulation, starting with 200 &#181;l cell pellets. Presence of RA in cell culture leads to ESC differentiation. From the cell pellet, about 50 - 100 &#181;g of histones were extracted, which is more than sufficient to perform multiple LC-MS injections of histone peptides. After derivatization, digestion, and desalting, the samples were loaded onto a 75 &#181;m x 15 cm C18 column (particle diameter 3 &#181;m, pore size 300 &#197;) in serial mode with a high-performance liquid nano chromatography system with microfluidic chips coupled to a hybrid linear trap quadrupole &#8211; orbitrap mass spectrometer. MS acquisition was performed using DIA. In parallel, samples were also analyzed with a DDA method using a nano-flow UHPLC coupled to a hybrid ion trap-orbitrap mass spectrometer (data not shown). In each cycle, one full MS orbitrap detection was performed with the scan range of 290 to 1,400 m/z, a resolution of 60,000 (at 200 m/z) and AGC of 106. Then, data dependent acquisition mode was applied with a dynamic exclusion of 30 sec. MS/MS scans were followed on parent ions from the most intense ones. Ions with a charge state of one were excluded from MS/MS. An isolation window of 2 m/z was used. Ions were fragmented using collision induced dissociation (CID) with collision energy of 35%. Ion trap detection was used with normal scan range mode and normal scan rate with AGC of 104.
Raw MS data were analyzed adopting software for the extraction of precursor and fragment ion chromatograms, namely Skyline23 and EpiProfile22. EpiProfile has been optimized for histone peptides, as it integrates intelligent peak area extraction due to previous knowledge of peptide retention time. On the other hand, Skyline is optimized for DIA analyses, and thus the DIA figures displayed (Figures 4 and 5A) are screenshots from this software. From the extracted ion chromatogram, the area under the curve is retrieved, and this is used to estimate the abundance of each peptide. The area of the chromatographic peak was calculated for the [M + H]+, [M + 2H]2+, and [M + 3H]3+ ions of the same peptide, even though in most cases the [M + 2H]2+ was the prevalent form. This provides the raw abundance of a given modified form of a peptide. In order to achieve the relative abundance of PTMs, the sum of all different modified forms of a histone peptide was considered as 100%, and the area of the particular peptide was divided by the total area for that histone peptide in all of its modified forms.
Histone peptides are present in a variety of isobaric forms (Figure 5). Isobaric peptides, e.g., K18ac and K23ac, can only be quantified at the MS/MS level, where their unique fragment ions are used to determine the ratio of the isobaric species (Figure 5A and 5B). This ratio is used to divide the area of the chromatographic peak between the two species. When using DDA, these isobaric forms were included in a list of targeted masses, because these peptides need to be selected for fragmentation through their entire elution, which would not occur in a standard DDA experiment. The discrimination of the relative abundance of the isobaric species is then performed by monitoring the elution profile of the fragment ions. On the other hand, DIA type of acquisition does not require any inclusion list. However, this type of acquisition method is not compatible with traditional database searching, and thus might prevent the discovery of unknown modified peptides.
Lysine acetylation (+ 42.011 Da) was discriminated from the nearly isobaric trimethylation (+ 42.047 Da) by using high resolution MS acquisition (&#62; 30,000). Moreover, acetylation is more hydrophobic than trimethylation, leading to elution of acetylated peptides later than the respective trimethylated ones. The unmodified form of the same peptide elutes even later, due to the fact that the lysine is propionylated. In summary, the order of hydrophobicity for a peptide with one modifiable site is di- and trimethylated &#60; acetylated &#60; unmodified (propionylated) &#60; monomethylated (propionylated).
hESCs showed a clear reduction of acetylated peptides when stimulated for differentiation (Figure 6A and 6B). This was not surprising, as previous results reported higher acetylation in ESCs as compared to differentiating ones25, reflecting the generally permissive nature of the pluripotent chromatin. By focusing on histone H3, 35 different modified forms were quantified (Figure 6C). However, all histone proteoforms that can be investigated with this approach are more than 200, including all histone variants and low abundance modifications (data not shown). Moreover, our analysis showed that high reproducibility can be obtained between technical replicates, as evidenced by the small size of the error bars (representing &#177; standard deviation). Taken together, this section describes how to extract the relative abundance of histone modified peptides using nLC-MS data.
<img alt="Figure 1" src="/files/ftp_upload/54112/54112fig1.jpg" /> 
Figure 1: Workflow for Bottom-up MS/MS Histone Analysis. The ten steps for histone analysis are shown, including an estimation of the time required for each step. The section number is given in parenthesis as present in the manuscript. Section 5, describing sample fractionation to isolate the various histone variants, can be omitted unless there is a need for highly sensitive analysis of a given variant. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54112/54112fig2.jpg" /> 
Figure 2: Reversed-Phase High Flow LC for Histone Variant Fractionation and Coomassie Gel. (A) LC-UV chromatogram representing intact histone separation. Histone H3 variants can be discriminated from one another according to their elution time. Fractions can be collected either manually or using an automated fraction collector. (B) Coomassie gel of three replicates of histone purification. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54112/54112fig3.jpg" /> 
Figure 3: Making of Stage-tipping Plug. With a P1000 pipette tip, punch a disk made of C18 material from a solid phase extraction disk (second panel). The minidisk will stick in the tip (middle panel), so that it can be pushed out into a smaller P100/200 pipette tip using any kind of small capillary. In this example, we used a 700 &#181;m external diameter fused silica tubing. The minidisk should be pushed to the bottom of the P100/200 pipette tip until it cannot go any further (last panel). The stage tip is ready for histone desalting, as it has sufficient capacity to retain enough sample material for numerous replicates. Specifically, one minidisk is enough for 15 - 20 &#181;g of sample. If more sample is required, multiple disks can be packed on one another. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54112/54112fig4.jpg" /> 
Figure 4: Schematic Representation of DDA and DIA Methods. When using DDA, the MS scan cycle is characterized by sequential selection of precursor ions for MS/MS fragmentation according to their intensity and charge state. Once a precursor ion has been fragmented it is placed into an exclusion list to avoid repetitive selection of the same peptide, so that the MS can &#34;dig&#34; into less abundant signals. This acquisition method is the technique of choice in proteomics for discovery mode. Quantification is achieved by integrating the full scan signal of a given ion next to the identified MS/MS spectrum. In DIA, the entire m/z range is fragmented at every scan cycle. This approach is less suitable for discovery mode, but it produces a chromatographic profile of all ions, precursors and products. This leads to more confident quantification and discrimination of isobaric forms. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54112/54112fig5.jpg" /> 
Figure 5: Quantification of Isobaric Peptides. (A) Example of two isobaric peptides commonly abundant in histone analysis. The extracted ion chromatogram (XIC) of their precursor mass and relative isotopes (above) is identical. However, the XIC of the product ions (below) allows for discrimination of the two isobaric forms. Notably, only unique fragment ions should be used to estimate the relative abundance of the two species. (B) Representation of the unique fragment ions for the two described peptides (highlighted in red). (C) List of the commonly analyzed peptides in Homo sapiens having at least one isobaric equivalent. Sequence variants between the listed histone peptides are indicated. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54112/54112fig6.jpg" /> 
Figure 6: Representative Results of Human Embryonic Stem Cells with and without Retinoic Acid Treatment. (A) Relative quantification of the histone H3 peptide KQLATKAAR (aa 18 - 26) in all of its modified proteoforms. The relative abundance was estimated using all proteoforms as 100% (the relative percentage of the unmodified peptide is not shown). (B) Relative quantification of the histone H3 peptide KSTGGKAPR (aa 9 - 17). (C) Relative abundance of detected peptides for canonical histone H3 with and without cell treatment with retinoic acid. The figure indicates in which of the two treatments the given modifications are more abundant (&#62; 50%). Overall, we demonstrate that histone H3 acetylation decreases in most of the lysine residues upon induction of cell differentiation. <a href="https://www.jove.com/files/ftp_upload/54112/54112fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Solution #</td>
			<td colspan="7">Composition</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="7">Nuclear Isolation Buffer (NIB) stock is made as follows and stored frozen as 100 ml aliquots at -20 &#176;C; thawed NIB can be stored at 4 &#176;C&#160; for few weeks: 15 mM Tris, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 250 mM sucrose. The pH of the buffer is adjusted to 7.5 with HCl.&#160;</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="7">Protease inhibitors (add fresh to buffers prior to use): 1 M Dithiothreitol (DTT) in ddH2O (1,000x); 200 mM AEBSF in ddH2O (400x)</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="7">phosphatase inhibitor (add fresh to buffers prior to use): 2.5 &#181;M Microcystin in 100% ethanol (500x)</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="7">HDAC inhibitor (add fresh to buffers prior to use): 5 M Sodium butyrate, made by titration of 5 M butyric acid using NaOH to pH 7.0 (500x)</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="7">NP-40 Alternative: 10% v/v in ddH2O</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="7">0.2 M H2SO4 in ddH2O</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="7">Trichloroacetic acid (TCA): 100% w/v in ddH2O</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="7">Acetone+0.1% Hydrochloric acid (HCl): 0.1% v/v HCl in acetone</td>
		</tr>
	</tbody>
</table>
Table 1. Solutions.

Watch this Scientific Journal Video about Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis at JoVE

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

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 ...

complete-workflow-for-analysis-histone-post-translational

Research

JoVE Journal

Biology

28.8K Views.  University of Pennsylvania. 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.

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

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

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 ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...