Histone Purification Combined with High‐Resolution Mass Spectrometry to Examine Histone Post‐Translational Modifications and Histone Variants in Caenorhabditis elegans

Abstract Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA‐related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post‐translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)−based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co‐occurring PTMs and the discovery of novel PTMs become feasible. The success of MS‐based approaches relies substantially on obtaining pure and well‐preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom‐up MS, and then evaluate the resulting data by a C. elegans−adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large‐scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom‐up mass spectrometry analysis of histone PTMs and histone variants


INTRODUCTION
This article provides a detailed set of protocols for the efficient extraction and purification of histone proteins from whole animals of the model organism Caenorhabditis elegans, achieving unprecedented purity at sufficient yield for comprehensive analysis of histone post-translational modifications (PTMs) and histone variants by bottom-up mass spectrometry. First, we describe how to grow this popular nematode model organism to a large scale by culturing it in liquid medium (Basic Protocol 1). Then, we present a method to isolate and lyse nuclei from the harvested animals, and to eventually purify nuclear histones by cation-exchange chromatography (Basic Protocol 2). This method achieves sufficient purity so that the obtained histones can be used directly for mass spectrometric analysis, avoiding any additional purification steps (e.g., by SDS-PAGE or HPLC) that would otherwise lower the yield. Quantification of histone PTMs and histone variants is then achieved by bottom-up mass spectrometry and subsequent analyses using a C. elegans−adapted version of EpiProfile 2.0, a recently developed software to analyze histone PTMs from MS measurements (Yuan et al., 2015(Yuan et al., , 2018) (Basic Protocol 3). Hereafter, we will refer to our C. elegans−adapted version of this software as EpiProfile 2.0-Ce. Figure 1 depicts a schematic workflow of the procedure in this article, and Tables 1 and 2 summarize all the histone PTMs and their combinations, as well as the histone variants that EpiProfile 2.0-Ce quantifies. To demonstrate the utility of our method, we determine the histone PTM composition of three C. elegans strains that each age at a different rate due to different levels of signaling through the insulin/IGF signaling (IIS) pathway and its downstream transcription factor DAF-16/FOXO. It has recently been reported that aging is associated with specific changes in histone PTM abundance (Booth & Brunet, 2016;Zhou, Sen, Lin, & Riedel, 2018). Using our method, we identify several expected but also some new histone PTMs whose abundance differs between these three strains.  The actual required amount of animals will depend on the purpose of the experiment. Normally, 0.5−1 million animals are sufficient to study most of the histone PTMs in adult wild-type animals. However, if one would like to detect particularly rare PTMs or analyze a C. elegans mutant that bears lower histone content, a higher starting amount is required. Here, it is important to know that in adult C. elegans, the tissue contributing the most to histones is the germ line. Thus, any mutant that lacks or has an impaired germline may need to be grown on an increased scale to ultimately yield the same histone amount as wild-type animals.

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Figure 1 Workflow for the extraction, purification, and mass spectrometric analysis of histone PTMs from C. elegans. Synchronized worms are grown on a large scale in liquid culture and eventually harvested (Basic Protocol 1). Upon worm lysis by mechanical methods, nuclei are isolated and histones extracted using acid (H 2 SO 4 ). To achieve high histone purity for mass spectrometry, crude histone extracts are subjected to ion-exchange chromatography. Histone yield and purity are verified by BCA protein quantification and by SDS-PAGE followed by Coomassie staining (Basic Protocol 2). Finally, histone samples are prepared for bottom-up mass spectrometry by propionylation and tryptic digestion. After sample desalting, nanoLC-MS/MS is performed and data analyzed by the software EpiProfile 2.0-Ce (Basic Protocol 3).
In order to prevent worms from digging into the agar, which would cause starvation and thereby affect the animals'physiology, avoid incubating them for extended periods of time on 150-mm diameter plates (2 days at maximum).
2. Wash worms off the plates using 12 ml of M9 buffer.
3. Collect worms in a 15-ml tube and centrifuge 1 min at 1500 × g to pellet the worms. Discard the supernatant.
4. Repeat this harvesting/washing step until all worms have been removed from the plate and the supernatant from step 3 is clear (approximately three washes).
5. After the final wash, resuspend worms in 7.5 ml sterile M9 buffer.
If your worm pellet is bigger than 1 ml, split the sample into multiple tubes and add M9 to 7.5 ml final volume, to bring the pellet volume below 1 ml.
6. Add 2.5 ml of fresh hypochlorite solution.
8. Examine under a stereomicroscope to determine if most worm corpses have disappeared and many released eggs can be seen.
9. Repeat steps 7 and 8 until all eggs have been released and barely any corpses remain. Overall, this bleaching (steps 7 to 9) will take approximately 6 min.
10. Centrifuge the tube 1 min at 1500 × g to pellet the released eggs.
11. Carefully take off the supernatant and discard it.
12. Wash eggs at least five times, each time with 10 ml M9 buffer, centrifuging 1 min at 1500 × g between washes.

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Current Protocols in Protein Science Make sure to go through the first two washes quickly, as a toxic amount of bleach will still remain during the first washes.
14. Rotate the tube containing the eggs for 1 day at room temperature or 2 days at 15°C.

Large-scale growth of C. elegans in liquid culture
15. Check that there are no unhatched eggs left, and determine the concentration of L1s (first stage larvae) by placing three spots of 5 or 10 μl of the C. elegans suspension on an NGM plate and counting the L1 larvae under a stereomicroscope.
16. For each sample/condition, prepare a 2-L Erlenmeyer flask containing 1 L of S-Complete Medium.
17. Seed the 1 L of S-Complete Medium with ∼80 ml of 50× OP50-1 bacteria. If you would like to analyze adult stages beyond day 1 of adulthood, the production of progeny will interfere with the experiment, leading to overcrowding of the culture with animals from the next generation. To avoid this, conditionally sterile C. elegans mutants (e.g., the strain CF512) can be used, or a sterilizing drug can be added prior to progeny production [e.g., 50 μM 5-fluoro-2 -deoxyuridine (FUDR)].

Harvest worms at a defined time point
From this point on, use cold buffers and cold centrifuges, and work on ice or in a cold room.
If the worm culture contains undesired eggs or worms in early larval stages, 40-μm cell strainers can be used to remove them by filtering the culture before proceeding to the next steps.

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Current Protocols in Protein Science 24. Allow the worms to settle for 10 min on ice, then centrifuge 3 min at 1500 × g, 4°C.
Discard the supernatant.
If you only have a limited number of 500-ml conical centrifuge tubes available, you can harvest more than 500 ml of culture in the same tube by repeating steps 23-24. 25. Transfer the worms to 50-ml centrifuge tubes.
26. Wash the worms with M9 buffer and collect them by spinning them down 2 min at 1500 × g, 4°C. Repeat this wash step until the supernatant is cleared of bacteria (approximately three washes).
29. Take off supernatant to just above the worm pellet.
30. Mix one volume of worm pellet with two volumes of ice-cold NIB + buffer.
31. Write down the total volume of the worm suspension (worm pellet plus NIB + buffer; used later in Basic Protocol 2) and then snap-freeze the worms by slowly dripping the suspension into a bucket filled with liquid nitrogen (to make beads).
PAUSE POINT: Frozen worm beads can be stored at −80°C for the long term.

NUCLEAR PREPARATION, HISTONE EXTRACTION, AND HISTONE PURIFICATION
The following acid extraction protocol is optimized to universally isolate all histone proteins and their variants. However, for the more efficient extraction of certain histone variants with unique biochemical and biophysical properties, other methods might be considered. For instance, extraction by diluted perchloric acid is a powerful tool to specifically extract more unstructured histone H1 variants (Zougman & Wiśniewski, 2006).
After acid extraction, we achieve histone purification and enrichment by subjecting the extracted material to ion-exchange chromatography. Histone proteins are rich in basic amino acid residues, and are thus positively charged. In vivo, this promotes their interaction with the negatively charged phosphate backbone of DNA. In vitro, we can make use of this property. Due to their positive charge, histones have a high affinity for cationexchange resins, which allows for their efficient separation from other contaminants (see Rodriguez-Collazo, Leuba, & Zlatanova, 2009).

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Scientific MegaFuge 40R) Benchtop refrigerated microcentrifuge (e.g., Eppendorf 5424R) Large refrigerated centrifuge with rotor for round-bottom tubes (e.g., Beckman Avanti with JA-15.50 rotor) 13-ml round-bottom centrifuge tubes with lid (Sarstedt, 55.518 and 65.816) Rotary mixer 1.5-ml microcentrifuge tubes Poly-Prep chromatography columns (Bio-Rad, 7311550) pH test paper 50-ml centrifuge tubes Additional reagents and equipment for protein assay (see Current Protocols article: Olson & Markwell, 2007) and gel electrophoresis (see Current Protocols article: Gallagher, 2012) Freeze-grinding and nuclear preparation 1. To fragment the frozen worms, freeze-grind the worm beads from Basic Protocol 1 using a cryogenic grinder. Using this device, grind until the worms are entirely Millan-Ariño et al.

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Current Protocols in Protein Science destroyed and no worm fragments remain visible upon checking a small amount of the ground material under a stereomicroscope after thawing. 3. Transfer the sample to a pre-chilled Dounce homogenizer. Use either a 7-ml or 15-ml Dounce homogenizer depending on the sample volume.
4. Continue the lysis of the animals and their cells using 50 strokes in the Dounce homogenizer on ice (10 strokes with pestle A and 40 strokes with pestle B).
If the sample volume exceeds the capacity supported by the Dounce homogenizer, then conduct the Dounce homogenization in batches.
5. Transfer the sample to a pre-chilled 15-ml tube. Split into several tubes if needed.
6. Take 50 μl of the sample and keep it at −80°C for later analysis.
This is sample A: whole worm extract; see Figure 2A for SDS-PAGE and Coomassie staining of whole worm extract starting material.
7. Spin the sample for 2 min at 200 × g, 4°C, to get rid of any debris.
8. Transfer the supernatant to a pre-chilled 13-ml round-bottom tube. Split into several tubes if needed. Keep pellets at −80°C for later optional analysis. This is sample B: debris pellet.
10. Remove the supernatant containing the cytoplasmic fraction and store it at −80°C for later optional analysis.
This is sample C: cytoplasm.
11. Disrupt the pellet containing the intact nuclei by resuspending in 1.67 ml of Buffer H + per 1 ml of total volume of worm suspension that was snap-frozen (see last step of Basic Protocol 1).  13. Transfer the homogenate to a 13-ml round-bottom tube and rotate it for 30 min at 4°C on a rotary mixer.
14. Spin the sample for 10 min at 10,000 × g, 4°C, to pellet the chromatin.
15. Collect the supernatant and store it at −80°C for later optional analysis.
This is sample D: nucleoplasm.

Acid extraction of histones
16. Wash the chromatin pellet three times in 1 to 3 ml B-Hypo-Lys Buffer freshly supplemented with inhibitors: 500 μM AEBSF 5 nM microcystin 10 mM sodium butyrate 1 mM DTT 1× cOmplete TM protease inhibitor cocktail. Mix well until the pellet is dissolved. For easier pellet resuspension, resuspend first with 100 μl and then add the remaining buffer.

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Current Protocols in Protein Science 18. Collect the supernatant and store it at −80°C for later optional analysis. This is sample E: chromatin wash.
19. Re-spin for 1 min and take off any remaining supernatant after the last wash. 20. Resuspend the pellet in 1 ml 0.4 N H 2 SO 4 and transfer the sample to a 1.5-ml microcentrifuge tube.
Mix well until the pellet is dissolved. For easier pellet resuspension, resuspend first with 100 μl and then add the remaining acid.
21. Rotate on a rotary mixer overnight at 4°C.

Histone purification by cation-exchange chromatography Column preparation
For your convenience, you can prepare the resin and column already on the day before the histone purification. The prepared column should be stored at 4°C with the top and bottom of the column properly closed to avoid drying of the resin. Be careful not to disturb the resin bed when adding liquids to the column during this and the following steps.
29. Repeat step 28 two more times for a total of three washes. 31. Repeat step 30 two more times for a total of three washes.
32. Close also the top of the column and store it at 4°C until the histone sample is added.

Histone purification
Perform all subsequent steps on ice or at 4°C to avoid enzymatic activities that may alter histone PTMs.
33. After the overnight incubation, spin down the acid extract (from step 21) for 5 min at 14,000 × g, 4°C.
34. Collect the supernatant in a 15-ml centrifuge tube. Keep the pellet at −80°C for later optional analysis. This is sample F: acid extraction pellet.

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Current Protocols in Protein Science 35. Also take 50 μl of the supernatant and store at −80°C for later optional analysis. 44. Take the bottom cap off again and add the remaining extract.
45. Allow the extract to pass through the column by gravity flow.
If here or at any later stage the gravity flow rate becomes unbearably low, you can consider centrifuging the column inside a 50-ml centrifuge tube for 3 min at 100 × g, 4°C, as done in step 41.

Keep the flow-through for later optional analysis.
This is sample I: 1 st column flow-through.
47. Wash the SP-resin with at least 10 ml histone wash buffer by gravity flow.

Keep flow-through for later optional analysis.
This is sample J: 2 nd column flow-through.
49. Add 1 ml histone elution buffer to the column and collect the eluate. Repeat this step two more times and collect each eluate into a different tube (eluate 1, eluate 2, and eluate 3).
51. Transfer supernatants to fresh tubes. Make sure not to touch any pellets; it is better to leave a bit of the supernatant behind rather than disturb the pellet.
52. Store pellets at −80°C for later optional analysis.

Quality control of the histone extraction by SDS-PAGE and Coomassie staining
To assess the purity of the extracted histones before proceeding to MS analysis, samples are subjected to SDS-PAGE and Coomassie staining. Core histones are low-molecularweight proteins, migrating between ∼11 and 15 kDa. High-percentage (15%) or gradient (4%-20%) Tris-glycine gels are recommended to efficiently resolve them. Starting material, purified histones, and any optional samples collected throughout the purification can be run together on the same gel. See Figure 2B for an example of how the final histone sample purity compares with the original starting material ( Fig. 2A

BOTTOM-UP MASS SPECTROMETRY ANALYSIS OF HISTONE PTMS AND HISTONE VARIANTS
In this protocol, the histone samples from Basic Protocol 2 are prepared and analyzed by MS. We use a bottom-up MS approach, where we first cleave the histones by trypsin into small peptides (5-15 amino acids in length), separate them by nano-scale reversed-phase chromatography (nanoLC), and then quantify them by tandem MS (MS/MS) (Karch, Sidoli, & Garcia, 2016;Sidoli, Bhanu, Karch, Wang, & Garcia, 2016a). However, our approach differs from standard protocols by the addition of two alkylation steps. First, we know that trypsin cuts after lysine and arginine residues and that histones are very basic, containing many of these amino acids. As a consequence, trypsin would normally cleave histones into peptides that are too small for optimal MS/MS analysis (Garcia et al., 2007). To address this, we use propionic acid to alkylate the ζ -amino groups of unmodified or monomethylated lysine residues, which prevents tryptic digestion at those sites, resulting in peptides that are longer and much more suitable. Second, once tryptic digestion is complete, we again use propionic acid, this time to alkylate each peptide's newly generated N-terminus. This aids the separation of peptides during nanoLC and further improves MS/MS results. To learn more, also about alternative MS approaches to study histone PTMs and variants, please consult Karch et al. (2016).
Once the MS/MS data have been collected, their analysis is challenging, most importantly because histones carry so many PTMs that can occur alone or in combination with others in a given peptide. This results in a large number of possible peptides that need to be distinguished from others. Another challenge is the existence of isobaric peptides that have the same m/z and similar elution times. Thus, we have developed a software application called EpiProfile (Yuan et al., 2015(Yuan et al., , 2018 to overcome these issues and provide a straightforward solution for the analysis of histone MS/MS data. Here, we use a C. elegans−adapted version of this software, called EpiProfile 2.0-Ce.

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Propionylation of lysine residues
1. Use ∼20 μg of purified histones from Basic Protocol 2, dry samples down in a vacuum centrifuge and resuspend them in 25 μl of 50 mM NH 4 HCO 3 , pH 8.0. Adjust sample concentration with concentrated NH 4 HCO 3 if samples were in Milli-Q water, or dry samples down in a vacuum centrifuge and resuspend with NH 4 HCO 3 if samples were in NaHCO 3 .
2. Use a P10 pipette and spot some sample onto pH paper, to ensure that the pH is around 8.0. Otherwise adjust it by adding glacial acetic acid or powdered NH 4 HCO 3 .
CAUTION: Use fume hood when working with propionic anhydride.
4. Add 10 μl of propionylation reagent to the sample and vortex briefly.
5. Check the pH with pH paper. It now should be between 4 and 6. Immediately add 3-7 μl NH 4 OH to increase the pH to ∼8.
Never allow the pH to increase above 10, as other residues such as serine can be propionylated at high pH.
7. Dry down to less than 5 μl in a vacuum centrifuge. This might take several minutes.
8. Repeat steps 1-7 once again with fresh reagents to ensure that >95% of the reactions are complete.
12. Quench trypsin by freezing at −80°C or by adding 5 μl glacial acetic acid to lower the pH to ∼4.
13. Dry sample down to 5 μl in a vacuum centrifuge.
PAUSE POINT: Sample can be stored at −80°C.

Propionylation of the N-termini of the tryptic peptides
14. Add 15 μl 100 mM NH 4 HCO 3 , pH 8.0, for a final volume of 20 μl and confirm that the pH has remained around 8.
16. Dry sample down to less than 5 μl in a vacuum centrifuge. This might take several minutes.
17. Add 200 μl of wash buffer to the sample. Check that the pH is below 4.0 with pH paper. If needed, adjust the pH by adding glacial acetic acid.
PAUSE POINT: Sample can be stored at −80°C.

Sample desalting with C 18 Stage-Tip
This step will remove salt from the digested sample, which otherwise may interfere with the nanoLC-MS/MS analysis.

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Current Protocols in Protein Science 18. Construct home-made C 18 columns or stage-tips as described in Sidoli et al. (2016a). Briefly, punch a C18 material disk with a P1000 pipette tip previously cut to make the hole larger, push the minidisk out of the P1000 tip with the help of a fused silica capillary, and place it firmly to the bottom of a P100/200 pipette tip. If desalting more than 25 μg of protein, use two C 18 minidisks in the same P100/P200 tip.
19. Place the column or stage-tips in a 1.5-ml or 2-ml microcentrifuge tube. Use a centrifuge adapter to hold the column or tip in place.
20. Activate the resin by adding 50 μl methanol to the column/tip and spinning 30-60 s at 500 × g.

MS acquisition
In this protocol, bottom-up MS data are acquired through a combination of datadependent acquisition (DDA) with targeted MS2 scans to distinguish coeluting isobaric peptides (see Table 3). To eliminate the need for targeting coeluting isobaric peptides, data-independent acquisition (DIA) methods can be a good alternative to DDA, although they are not recommended for identifying unknown peptides. For details on how to perform DIA consult Karch et al. (2016).

Data analysis using EpiProfile 2.0-Ce
Although quantification of histone peptides can be achieved manually, e.g., by using the Xcalibur QualBrowser (Thermo) or by using the freeware Skyline (MacLean et al., 2010), we recommend the use of EpiProfile. This software has been specifically developed for the quantification of histone PTMs and variants from MS/MS data, including both DDA and DIA approaches (Yuan et al., 2015(Yuan et al., , 2018. EpiProfile is a freely available Matlab-based automated tool that reads raw data files and outputs tables of quantified histone peptides (see Table 4 for an example), layouts (MS1 elution profiles, Fig. 3A), and annotated MS/MS spectra used for identification. EpiProfile is very easy to use and can quantify coeluting isobaric peptides ( Fig. 3B and 3C). Since its original release, EpiProfile has been adapted for the study of histones in different organisms including humans, mice, C. elegans, and yeast. We recommend consulting previous publications if you would like to obtain a deeper understanding of EpiProfile (Yuan et al., 2015(Yuan et al., , 2018.

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Coomassie solution
Prepare in Milli-Q water: Store up to several months at room temperature

Destaining solution
Prepare in Milli-Q water: 10% (v/v) acetic acid 10% (v/v) 2-propanol Store up to several months at room temperature

M9 buffer (2 L)
6 g KH 2 PO 4 12 g Na 2 HPO 4 10 g NaCl 2 L sterile Milli-Q water or equivalent Sterilize by autoclaving Store up to several months at room temperature

Nuclear isolation buffer (NIB) and NIB + buffer
Prepare NIB stock: 25 mM HEPES, pH 7.5 0.5 M sucrose 25 mM KCI 10 mM MgCl 2 0.1 mM EDTA, pH 8.0 Store NIB stock with the above ingredients up to several months at 4°C Millan-Ariño et al.

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S-Basal
5.85 g NaCl 1 g K 2 HPO 4 6 g KH 2 PO 4 1 ml 5 mg/ml cholesterol in ethanol Milli-Q water (or equivalent) to 1 L Sterilize by autoclaving Store up to several months at room temperature
Similar to other proteins in the cell, histones can be post-translationally modified by different enzymes that deposit or remove chemical groups at individual amino acid residues. Most of these PTMs occur in histones' exposed N-and C-terminal tails, but some also exist in their globular domains (Kouzarides, 2007). Methylation, acetylation, and phosphorylation are the most common and best-studied histone PTMs. Nevertheless, there is increasing evidence for other modifications (i.e., ADP-ribosylation, crotonylation, SUMOylation, or citrullination) that are now studied as well . Canonical histones are expressed during S phase of the cell cycle, transcribed from multiple gene copies that are organized in clusters within the genome. During evolution, many histone variants emerged from these canonical histones, all of which have altered biochemical and biophysical properties due to minor sequence differences, and thereby have acquired specific functions needed in specific contexts (Millán-Ariño, Izquierdo-Bouldstridge, & Jordan, 2016;Zink & Hake, 2016). In contrast to canonical histones, these variants are typically encoded only from a single gene locus and expressed throughout the cell cycle. Dynamics of both histone PTMs and histone variants are associated with different physiological processes such as cellular differentiation or the aging process (Atlasi & Stunnenberg, 2017;Booth & Brunet, 2016), and their imbalance or dysregulation is tightly linked with many pathologies (Feinberg, Koldobskiy, & Göndör, 2016). This also makes them a potential target to treat such pathologies (Morel, Jeffery, Aspeslagh, Almouzni, & Postel-Vinay, 2019).
The conventional methods of studying histone PTMs via western blotting or Luminex approaches are limited by the availability of specific antibodies recognizing all these modified residues. Although a broad palette of antibodies is currently available from different vendors, good antibodies are not available for all PTMs, PTM combinations, and variants.

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Current Protocols in Protein Science Moreover, differences in histone amino acid sequence between species can further limit the availability of good antibodies for less commonly studied organisms. Mass spectrometry methods have recently helped to overcome these issues. They make it possible to study and quantify dozens of histone PTMs in a single experiment, to quantify combinatorial occurrences of PTMs within the same peptides (Karch, DeNizio, Black, & Garcia, 2013), and to obtain this information in any organism. New modifications can even be discovered (Britton, Gonzales-Cope, Zee, & Garcia, 2011). By now, several studies have described mass spectrometry methods to quantify histone PTMs and variants (Huang, Lin, Garcia, & Zhao, 2015). However, what is crucial for the success of all these approaches is the quality of the histone material to be analyzed. While from some species, e.g., human cells, histones can be obtained rather easily by mere acid extraction, in other species like C. elegans this can be very challenging. Particular effort needs to be exercised in order to obtain highpurity nuclear histone preparations at sufficient yield while preserving their PTMs.

Synchrony and reproducibility of C. elegans cultures
The methodology described in this article has been developed to study differences in histone PTMs or variant abundance between different C. elegans samples. Since the epigenome, and thus these marks, can drastically change depending on environmental influences, including food availability (Berger & Sassone-Corsi, 2016), or on the developmental stage of the organism (Sidoli, Vandamme, Salcini, & Jensen, 2016b), it is important to avoid any variability in the culturing conditions and to make sure that all samples are perfectly synchronized to the same developmental stage or age. Thus, the protocols provided here for C. elegans liquid culture should be strictly followed, and culturing conditions (i.e., temperature and food availability) should be carefully monitored and kept as constant as possible between replicates and throughout the experiment. In particular, we recommend using a temperature tracker, which is placed inside the shaking incubator and checked daily. Furthermore, the abundance of OP50-1 E. coli in the medium is checked daily by OD 600 measurement, to remain between 2 and 4 absorbance units. For this, 10 ml of culture are transferred to a 15-ml tube, the C. elegans are settled by gravity for 10 min, and then an aliquot of the supernatant is subjected to the OD measurement at 600 nm. Finally, the developmental stage of the animals and thus synchrony between samples and replicates should be checked daily during the first days of the experiment. Should any sample show delays in progressing through development, this delay should be quantified and the sample harvested at an accordingly later time.

Input material
The above set of protocols grows 0.5-1 million C. elegans and eventually recovers 20-30 μg of pure histones. However, sometimes more animals should be used. This is particularly true for certain strains, e.g., strains that have a retarded germline, for the analysis of animals from earlier developmental stages, or when you have an interest in quantifying particularly rare histone PTMs.

Achieving optimal yield
Besides increasing the amount of C. elegans used, there are a few additional points to consider in order to maximize your histone yield and preserve the histones' integrity and PTMs. First, it is important to maintain an optimal ratio of biological material and extraction/washing buffers throughout the extraction procedure. Closely follow the ratios indicated in the protocol and scale the buffer amounts depending on the amount of starting material that you use (worm pellet after harvesting). Second, for optimal histone extraction, animals need to be efficiently lysed. In C. elegans, this requires breakage of the cuticle, which we achieve by grinding in a cryogenic mill. Grinding results should be checked under a microscope. Upon optimal grinding, all animals should be broken. Ideally, no worm structures should be visible anymore. Further homogenization and lysis is achieved by a Dounce homogenizer. Make sure to use a Dounce homogenizer appropriate for the sample volume and with the correct pestle type.

Histone preservation
Proteins and their PTMs are more stable at lower temperatures, when proteases or enzymes that alter their PTMs tend to be less active. Thus, the histone extraction and purification should always be performed on ice or inside a cold room. For similar reasons, we recommend storing all collected samples at −80°C rather than 4°C or −20°C. It is furthermore important to use protease inhibitors Millan-Ariño et al.

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Current Protocols in Protein Science and stabilizing agents during the extraction procedure. The choice of stabilizing agents may be adjusted, depending on the histone modifications that you are interested in. In the protocols presented here, microcystin is used to preserve phosphorylations and sodium butyrate is used to preserve acetylations.

Ion-exchange chromatography
One of the main benefits of the protocols described above is the purity of the resulting histone samples. This is achieved by incorporation of an ion-exchange chromatography step, which removes most non-histone contaminants. As the purity is so high, no further purification (e.g., gel-purification or HPLC) is required before trypsin digestion and nanoLC-MS/MS. However, errors in executing the ionexchange chromatography may lead to impurities that interfere with nanoLC-MS/MS. We therefore recommend always evaluating the purity of your final histone sample by analytical SDS-PAGE and Coomassie staining. If substantial contamination from non-histone proteins can be seen, further purification by ion-exchange chromatography or gel purification should be considered; for future purifications, the ion-exchange chromatography should be conducted more carefully. Most importantly, make sure to properly neutralize the acid-extracted samples to a pH of 7-8 before loading them onto the column.

Histone alkylation and digestion
For a good analysis by MS/MS, histones should be optimally propionylated and the tryptic digestion should go to completion. To achieve this, multiple measures should be taken. In the purification protocol's final precipitation step, make sure to properly wash the histone pellet with acetone as described. Any traces of acid that remain with the sample would critically interfere with derivatization and digestion. We also suggest performing the propionylation quickly, not treating too many samples at the same time. And, before initiating the trypsin digestion, carefully confirm that the pH is ∼8.0. Issues with efficient alkylation and digestion would otherwise lead to low detection of the desired peptides.

Statistical Analysis
For the creation of the heat map in Figure 4, the following linear model was used: log2(mark) ∼ date + condition. This allowed us to account for batch effects and to obtain log2 fold-change values. To account for the high number of tests performed, we corrected our p-values for multiple testing using the False Discovery Rate method. Corrected p-values were computed using the q-value function from the R package q-value. Contrasts were manually set in order to compare different conditions. Clusters for the heatmap were computed by agglomerative hierarchical clustering using the hclust function. All analyses were performed in R version 3.6.3 (R Core Team, 2013) using the packages tidyverse (Wickham, 2016), magrittr (Milton Bache & Wickham, 2014), and Biobase (Huber et al., 2015).

Understanding Results
The quantification of histone posttranslational modifications in C. elegans can be a powerful tool to study how the epigenome contributes to specific aspects of this organism's physiology. To provide an example, we used it to determine, how histone PTMs differ between animals that age either quickly, normally, or slowly.
The nutrient-sensing insulin/IGF-like signaling (IIS) pathway is one of the best characterized signaling pathways that regulates the rate at which we age (Kimura, Tissenbaum, Liu, & Ruvkun, 1997;Tatar et al., 2001). When there are plenty of nutrients and IIS is high, aging occurs rather quickly, but when IIS is low, aging is slowed down. Mostly, the latter situation is caused by a transcription factor called DAF-16/FOXO. This transcription factor is negatively regulated by IIS and drives the expression of many aging-preventive genes (Murphy et al., 2003;Zhou et al., 2018).
For this study, we chose wild-type C. elegans, which age at a normal rate, a hypomorph of the insulin/IGF-like receptor daf-2 (daf-2(e1370ts)), which ages slowly and eventually reaches a very long lifespan, and a null mutant of daf-16 (daf-16(mu86lf)), which ages quickly and reaches only a very short lifespan. We brought these animals to scale, synchronized them, added 50 μM 5-fluoro-2deoxyuridine (FUDR) starting at the L4 stage to prevent progeny production, and eventually grew them until day 2 of adulthood. Animals were then harvested and lysed, and histones extracted, purified, and analyzed by nanoLC-MS/MS-all according to our protocols above. We then used EpiProfile 2.0-Ce to analyze the MS/MS data and quantify the histone PTMs and variants. EpiProfile uses previous knowledge about relative elution times for the identification and quantification of histone peptides [for details consult (Yuan et al., 2015(Yuan et al., , 2018]. For a given modification Figure 4 Bottom-up mass spectrometry uncovers differences in the relative abundance of histone PTMs between wild-type (N2), long-lived [daf-2(e1370ts)], and short-lived [daf-16(mu86lf)] C. elegans. (A) A heatmap of the changes in H3 and H4 PTM abundances for the comparisons of daf-2(e1370ts) versus wild-type and wild-type versus daf-16(mu86lf) animals. Significance of upor downregulated PTMs is indicated by asterisks (* = 0.1 < p > 0.05; ** = 0.05 < p > 0.01; *** = 0.01 < p > 0.001; **** = 0.001 < p > 0.0001; ***** = 0.0001 < p > 0; p-values were corrected for multiple testing by the False Discovery Rate method). (B) Combinatorial histone PTM landscape of the H3 18-23 peptide in the indicated C. elegans strains. Relative ratios of the unmodified and modified forms of the peptide are shown, as calculated by EpiProfile 2.0-Ce. Error bars represent the standard error across three MS runs. on a peptide backbone, EpiProfile calculates its relative abundance or ratio by dividing its area under the MS1 chromatogram by the sum of areas of all (un)modified forms of this same peptide backbone. This information is found in the output file histone_ratios.xlsx (see Table 4 for an example). It allows for the study of all possible modified forms of each peptide, and thus also the study of combinatorial histone PTMs and PTM switches within a given peptide. Besides that, EpiProfile also calculates the relative abundance of individual histone PTMs by summing the relative abundance of all peptides that contain a given modification. These data are found in the output file histone_ratios_single_PTMs. xlsx.
Relative quantification of PTMs on histones H3 and H4 (found in the histone_ ratios_single_PTMs.xlsx file) revealed substantial differences in the abundance of certain histone marks in slow-, normally, and fast-aging animals. The heat map in Figure 4A illustrates these differences on a log2-fold scale. Certain marks are Millan-Ariño et al.

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Current Protocols in Protein Science positively associated with animals that age more slowly (e.g., H3K18ac or H4K20ac), while others are rather negatively associated (e.g., H3K23me2/3 or H4K20me1). Our data indicate dynamic changes in histone residues H3K18, H3K23, and H4K20, from methylated to acetylated states in animals that age slowly. When looking at the relative abundance of all forms of the H3 18-26 peptide (found in the histone_ratios.xlsx file), we observed that the increasing acetylation at H3K18 coincides with acetylation of H3K23 of the same peptide (Fig. 4B). We conclude that H4K20ac and likely double-acetylation of H3 at K18 and K23 are marks associated with slow aging and longevity.

Time Considerations
The time to grow strains on a large scale before harvesting (Basic Protocol 1) is variable and depends on diverse parameters, e.g., the temperature, the strains used, the desired worm stage to harvest, etc. Whatever the case, the following should give you some guidance. The initial growth of C. elegans until they fill multiple 15-cm plates may take approximately 2 weeks. Egg preparation from gravid adults takes around 2 hr. Hatching of the embryos out of their eggshell and their arrest as L1 larvae is reached after 18 hr at room temperature or 2 days at 15°C. The growth in liquid culture will strongly depend on the stage of animals that you would like to harvest, and can take anywhere from 1 to many days. The harvesting of the liquid culture and freezing of the animals will take about 3 hr.
For Basic Protocol 2, the grinding procedure may take 1 to 2 hr. The resulting worm powder can be stored at −80°C or directly used for nuclear preparation and histone acid extraction. Those steps take 1 full day before leaving samples rotating overnight at 4°C with H 2 SO 4 . Another full day is required for the histone purification by cation-exchange chromatography and for the histone precipitation step. Quality control of histone samples by SDS-PAGE and Coomassie staining takes around 4 to 6 hr, including the time it takes to run the protein gels and the time for staining and destaining of the gel.
Finally, sample preparation for nanoLC-MS/MS and the actual nanoLC-MS/MS run (Basic Protocol 3) take 2-3 days. Data analysis by EpiProfile 2.0-Ce is highly automated and takes only a few minutes to run. However, additional analyses for data representation might take longer.