This material is based upon work supported by the National Science Foundation under Grant No. HRD-1547798 and Grant No. HRD-2111661. These NSF Grants were awarded to Florida International University as part of the Centers of Research Excellence in Science and Technology (CREST) Program. This is contribution number 1672 from the Institute of Environment, a Preeminent Program at Florida International University. Additional support was provided by the National Institute of Health under Grant No. R21AI135469 to Francisco Fernandez-Lima and Grant No. R01HD106051 to Benjamin A. Garcia, as well as by the National Science Foundation under Grant No. CHE-2127882 to Benjamin A. Garcia. The authors would like to acknowledge the initial support of Dr. Mario Gomez Hernandez during initial method developments.

NOTE: Histone samples were extracted using a method adapted from Bhanu et al. (2020)12.
1. Sample preparation
<ol>
	<li>Harvesting cultured cells
	<ol>
		<li>When cells are 80% confluent, ensure they are viable using trypan blue exclusion. 
		NOTE: A HeLa S3 cell line was used for these experiments, but this method can be applied to any cultured cells.</li>
		<li>Aspirate the media, then apply 5 mL of 1x phosphate-buffered saline (PBS) to each plate.</li>
		<li>Swirl the plate(s) to rinse all residual media, then aspirate PBS and apply 5 mL of 1x PBS.</li>
		<li>Gently separate the cells from the plate by scraping them with a disposable cell lifter.</li>
		<li>Transfer each cell suspension to a 15 mL conical tube.</li>
		<li>Pellet the cells by centrifuging at 800 x g for 5 min.</li>
		<li>Aspirate the PBS from the cell pellet.</li>
		<li>Proceed to histone extraction. 
		NOTE: Flash-freeze the cell pellet in liquid nitrogen if it cannot be processed immediately. Store the pellets at -80 &#176;C until ready to proceed.</li>
	</ol>
	</li>
	<li>Histone extraction
	<ol>
		<li>Estimate the volume of each cell pellet and mark the meniscus with a permanent marker.</li>
		<li>Prepare enough nuclear isolation buffer (NIB; 15 mM Tris-HCl (pH 7.5), 15 mM NaCl, 60 mM KCl, 5 mM MgCl2, 1 mM CaCl2, and 250 mM sucrose) for all the samples. Alternatively, if many samples need to be processed over time, make the buffer in bulk and store at 2-8 &#176;C for up to 6 months, or aliquot and freeze at -15 &#176;C to -25 &#176;C indefinitely by thawing only the amount necessary for each extraction. 
		NOTE: The buffer should remain clear during storage. If the buffer takes on a cloudy or otherwise abnormal appearance at any time, discard and prepare fresh buffer.</li>
		<li>Prepare 50 times the volume of the cell pellets of wash buffer and add inhibitors as follows (approximately 10 mL of wash buffer per 2 samples).
		<ol>
			<li>To prepare 10 mL of wash buffer, mix 10 mL of NIB, 30 &#181;L of 200 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride [AEBSF], 10 &#181;L of 1 M dithiothreitol [DTT], 20 &#181;L of 5 &#181;M microcystin, 20 &#181;L of 5 M sodium butyrate.</li>
		</ol>
		</li>
		<li>Remove 1/5 of the wash buffer to prepare the lysis buffer (1/5 volume from wash buffer, 0.3% NP-40 or NP-40 alternative). 
		NOTE: Do not use Triton-X 100 instead of NP-40 or NP-40 alternative, as it may be too abrasive for certain cell types.</li>
		<li>Wash the cell pellet thoroughly by suspending it in 5 columns of wash buffer and centrifuging at 800 x g for 5 min at 4 &#176;C. Complete this step twice, aspirating and discarding the supernatant between washes.</li>
		<li>Ensure the volume of the cell pellet is still marked with a permanent marker. Resuspend in 10 volumes of lysis buffer.</li>
		<li>Pipette-mix each pellet thoroughly to resuspend, then incubate for 15 min on ice.</li>
		<li>After 15 min, centrifuge at 800 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate and discard the supernatant. 
		NOTE: The pellet should reduce to &#8804; 1/2 the original pellet size (as indicated by the marker line). If the pellet has not reduced sufficiently, repeat the lysis procedure and include a gentle homogenization step using a pestle to break open the cells.</li>
		<li>Once lysis is complete, resuspend the pellet in 500 &#181;L of wash buffer, then centrifuge at 800 x g for 5 min at 4 &#176;C. Aspirate and discard the supernatant, then repeat the wash step once more to remove all traces of NP-40. 
		NOTE: At this point, the pellet consists of chromatin, which contains histones.</li>
		<li>Resuspend the pellet in 5 volumes (of the original cell pellet size) of 0.4 N H2SO4.</li>
		<li>Incubate for 2 h in a cold room or refrigerator using an agitator.</li>
		<li>After 2 h, centrifuge the sample(s) at 3400 x g for 5 min at 4 &#176;C. Do not discard the supernatant.</li>
		<li>Transfer the supernatant to new tubes, and spike 100% trichloroacetic acid (TCA) to 1/3 the volume of the contents (the final TCA concentration will be approximately 20%).</li>
		<li>Gently invert the tube and observe that the clear, colorless solution turns white and/or cloudy, indicating protein precipitation. 
		NOTE: For solutions with low histone concentrations, the protein precipitation may not be immediately noticeable, but the precipitate should be visible after the overnight incubation.</li>
		<li>Incubate without disturbance overnight (12-18 h) at 4 &#176;C to completely precipitate the histone proteins.</li>
		<li>The following day, centrifuge at 3400 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant, being careful not to touch the sides of the tube with the pipette tip. At this stage, the histones are deposited primarily as a film around the sides of the tube(s).</li>
		<li>Add 500 &#181;L of ice-cold acetone + 0.1% HCl (acid acetone) to each tube, using a glass Pasteur pipette, then gently invert the tube(s) several times. Arrange the samples in order (1, 2, 3, etc.) while doing this, as any errant acetone may remove the markings on the tubes. Centrifuge at 3400 x g for 5 min at 4 &#176;C and gently decant the supernatant.</li>
		<li>Repeat this rinsing step with 500 &#181;L of ice-cold 100% acetone, also with a glass Pasteur pipette. Centrifuge at 3400 x g for 5 min at 4 &#176;C and gently decant the supernatant.</li>
		<li>Leave the tubes open to dry at room temperature until the remaining acetone has evaporated.</li>
		<li>When dry, add 100 &#181;L of mass spectrometry (MS)-grade water to each tube. Use this droplet to swab all sides of the container to resuspend the entire histone film. Do this by pipetting the droplet onto the side of the tube and rotating it while pipetting up and down or dispensing half of the 100 &#181;L and using the tip to stir it all around. A combination of both methods works best. Histones are readily soluble in water and will be in the solution.</li>
		<li>After resuspending all samples, if there is any remaining white solid, sonicate in a bath at room temperature for 5 min.</li>
		<li>Centrifuge at 800 x g for 5 min at 4 &#176;C. Transfer the clear solution to fresh tubes. Discard any remaining insoluble pellet.</li>
		<li>Run an SDS-PAGE under reducing conditions to verify that the extraction is clean. 
		NOTE: Gels can be run using any appropriate concentration of polyacrylamide as long as it can differentiate proteins in the range of 10-20 kDa. See the Table of Materials&#160;for the gels used in this protocol.</li>
		<li>Perform a protein concentration assay (i.e., Bradford or BCA) to determine the total protein concentration.</li>
	</ol>
	</li>
	<li>Chemical derivatization (propionylation) of the lysine residues
	<ol>
		<li>Transfer 20 &#181;g of histones (determined by the protein assay) to a clean tube. Dry this sample down to &#60;5 &#181;L using a vacuum concentrator, then resuspend using 20 &#181;L of 100 mM ammonium bicarbonate (NH4CO3) (~1 &#181;g/&#181;L solution). Adjust the pH to ~8 using ammonium hydroxide if needed. 
		CAUTION: Do not use ammonium hydroxide (NH4OH) to resuspend, only to adjust the pH if necessary. Otherwise, proteins will denature and precipitate. 
		NOTE: To check the pH with minimal sample loss, use a pipette tip to dip in the sample and dab onto a pH strip. This testing procedure will be useful throughout the remaining sample preparation steps.</li>
		<li>Prepare the propionylation reagent by adding propionic anhydride to acetonitrile (ACN) in a 1:3 (v/v) ratio (i.e., to make 40 &#181;L of reagent, combine 10 &#181;L of propionic anhydride with 30 &#181;L of ACN). 
		NOTE: Traditionally, methanol or isopropanol have been used in preparation of a propionylation reagent. As propionylation is an amide formation reaction, a non-protonic solvent, like acetonitrile, is required to prevent unwanted side products and reactions, such as methyl propionyl ester, which results from using methanol. Only prepare enough propionylation reagent for up to 4 samples at a time so the reagent remains fresh. Use the reagent within 1-2 min of preparation. As the reagent sits, the propionic anhydride will react with any ambient moisture, and acetic acid will begin to form, which can change the effectiveness of the reagent and will change the pH of the histone solution once the reagent is added.</li>
		<li>Add propionylation reagent to each sample in a 1:4 (v/v) (i.e., for 20 &#181;L of histones, add 5 &#181;L of propionylation reagent).</li>
		<li>Quickly add 1:5 (v/v) NH4OH (i.e., add 4 &#181;L for 20 &#181;L of the histone solution) to re-establish the pH of the solution to ~8. If pH is still too low, add 1-2 &#181;L of NH4OH at a time until a pH of 8 is achieved. Typically, a 1:5 (v/v) ratio is adequate.</li>
		<li>Incubate the samples at room temperature for 15 min without disturbance.</li>
		<li>Repeat the propionylation reaction for no more than 3-4 samples per batch of propionylation reagent to ensure minimal acid formation.</li>
		<li>Repeat the propionylation procedure steps 1.3.2-1.3.5. A second round of propionylation ensures that &#62;95% of available lysines are derivatized.</li>
		<li>Dry the samples down to &#60;5 &#181;L using a vacuum concentrator. This will evaporate any unreacted propionylation reagent, acid products, and ammonia gas released from the NH4OH. If the samples dry out completely, this is fine, as no significant sample losses occur. 
		NOTE: Displace air in the propionic anhydride bottle with argon gas prior to storage to prevent the formation of acetic acid due to contact with ambient moisture remaining in the bottle.</li>
	</ol>
	</li>
	<li>Proteolytic digestion with trypsin
	<ol>
		<li>Resuspend histones in 100 mM NH4HCO3 to achieve a volume of 20 &#181;L, achieving an optimal concentration of 1 &#181;g/&#181;L. 
		NOTE: Sample solutions with concentrations lower than 1 &#181;g/&#181;L will result in decreased Trypsin efficiency.</li>
		<li>Add trypsin to histone samples at a 1:10 ratio (wt/wt) (i.e., add 2 &#181;L of 1 &#181;g/&#181;L solution of trypsin to 20 &#181;g of histones).</li>
		<li>Incubate reactions at 37 &#176;C for 6-8 h. Alternatively, incubate overnight (12-18 h) at room temperature.</li>
		<li>Stop the digestion by freezing at -80 &#176;C for at least 1 h. 
		NOTE: Do not use acid to quench the digestion reaction, as this will cause an unwanted drop in pH at this point in the procedure. The sample can be stored at -80 &#176;C until ready to proceed (Interim stopping point).</li>
	</ol>
	</li>
	<li>Chemical derivatization (propionylation) of peptide N-terminals
	<ol>
		<li>Dry the samples to &#60;5 &#181;L using a vacuum concentrator.</li>
		<li>Resuspend the samples up to 20 &#181;L (1 &#181;g/&#181;L) using 100 mM NH4HCO3.</li>
		<li>Repeat propionylation as before (step 1.3). 
		NOTE: It is normal that the samples take longer to dry at this step due to a higher aqueous: organic phase ratio.</li>
	</ol>
	</li>
	<li>Sample desalting with stage-tips
	<ol>
		<li>Resuspend or dilute samples with 50 &#181;L of MS-grade water + 0.1% TFA.</li>
		<li>Using an 11-G sample corer, punch 5 disks of C18 material from a solid phase extraction disk (punch all 5 disks before transferring to the pipette tip). Insert and ensure the disks are securely and evenly wedged at the bottom of a 200 &#181;L pipette tip (Figure 1). 
		NOTE: Use 15-G corer if desalting over 25 &#181;g of sample through a single stage-tip.</li>
		<li>Use a centrifuge adapter to hold the stage-tips in place in 1.5 mL or 2 mL microcentrifuge tubes. 
		NOTE: For the following centrifugation steps, use slow (400-500 x g) revolution at 4 &#176;C, for 1-2 min at a time; the solvents normally pass through the resin in less than 1 min, depending on how tightly the C18 material is packed into the tips.</li>
		<li>Rinse the resin by centrifuging with 50 &#181;L of 100% acetonitrile to activate the C18 material and remove potential contaminants. 
		NOTE: It may be easier to load solutions onto the stage-tips using gel-loading pipette tips. Once the C18 material has been activated, it is important not to allow the resin to dry out for the duration of the desalting procedure.</li>
		<li>Equilibrate the disk material with 80 &#181;L of MS-grade water + 0.1% TFA by centrifugation.</li>
		<li>Acidify the sample to pH 4 or lower using glacial acetic acid. Check the pH with pH strips as before to minimize sample loss.</li>
		<li>Load the entirety of the sample onto the resin disk by slow centrifugation.</li>
		<li>Wash the sample with 80 &#181;L of MS-grade water + 0.1% TFA by centrifugation.</li>
		<li>Elute the sample into a clean 1.5 mL tube by flushing 70 &#181;L of 75% acetonitrile and 0.5% acetic acid by slow centrifugation. Additional centrifugation time may be used to ensure the full sample volume is eluted from the stage-tip. It is okay if the resin dries out with the additional centrifugation time, as it is no longer needed past the elution of the sample.</li>
		<li>Dry each sample completely in a vacuum concentrator. 
		&#8203;NOTE: Sample(s) can be stored at -80 &#176;C until ready to proceed (Interim stopping point).</li>
		<li>For LC-MS/MS analysis, reconstitute the samples in a volume of Solvent A (0.1% formic acid) from the liquid chromatography (LC) protocol that gives the final concentration of 0.4 &#181;g/&#181;L (i.e., dissolve 20 &#181;g of histones in 50 &#181;L of Solvent A).</li>
	</ol>
	</li>
</ol>
2. TIMS software interface
<ol>
	<li>Select the Instrument tab and switch to Operate (verify that the instrument name becomes highlighted in green) (Figure 2).</li>
	<li>Verify TIMS parameters (Figure 2).</li>
	<li>Verify MS Settings (scan begin, scan end, ion polarity, scan mode) (Figure 2).</li>
	<li>Verify TIMS settings (mode, mobility start, mobility end, ramp time, accumulation time, duty cycle, ramp rate, MS rate, MS averaging, and autocalibration) (Figure 2).</li>
	<li>Go to the Source tab and activate the syringe option (Hamilton 500 &#181;L) only for the TuneMix calibration step (Figure 3)13.</li>
	<li>Go to the Calibration tab, click m/z, select Calibration Mode, and choose the mode (Enhanced Q, generally), zoom (+0.01%) STD Dev (0.24), and click Calibrate; when a score of 100% is achieved, accept (Figure 4).</li>
	<li>Go to the mobility tab and repeat the calibration process (Linear Mode, generally), detection range (+5%), width (0.1 Da), STD Dev (0.1855), then click calibrate; when you get a score &#8805; 98.5%, accept (Figure 5).</li>
	<li>Go to the method and select the method to be used; for this example, Proteomic_/2023-01-19-CF/20230119-Hela Control histone_prep_pasefDIA_1-24_1_451.d/451.m-TimsControl was selected (Figure 5).</li>
</ol>
3. LC-TIMS-PASEF-ToF MS/MS
<ol>
	<li>Use the typical nESI operating conditions: 4500 V capillary voltage, 800 V endplate offset, 3.0 bar nebulizer pressure, 10.0 L/min dry gas, 200 &#176;C dry heater, and 50 &#181;L/min injection flow rate.</li>
	<li>Use the typical MS settings: 6 eV collision energy, 1200 Vpp collision RF, 75 &#181;s transfer time, 5 &#181;s prepulse storage.</li>
	<li>Determine the drift gas flow using the pressure difference from the entrance funnel P1 and the exit funnel P2. Parallel accumulation-serial fragmentation (PASEF) occurs in the TIMS cell, accumulating all precursor ions simultaneously rather than individually. Precursor ions are then released in narrow ion peaks versus the normally much wider peaks (about 50 times shorter), increasing the signal-to-noise ratio while still separating co-eluting peptides via mobility14.</li>
	<li>Develop an LC-TIMS-ToF MS/MS method for analyzing proteolytic histone peptides. Couple a high-performance liquid chromatograph (HPLC) fitted with a C18 (300 &#197;, 5 &#181;m, 4.6 mm x 250 mm) column with a commercial TIMS-TOF MS instrument with proprietary PASEF technology. 
	NOTE: This column size was determined to provide good separation at both high and low pH for peptide mixtures, based on previously published works15,16,17.
	<ol>
		<li>Set the injection volume to 20 &#181;L (8 &#181;g) of sample and a 0.4 mL/min flow rate.</li>
		<li>Run a 60-min, non-linear LC gradient using water with 0.1% formic acid (Solvent A) and acetonitrile with 0.1% formic acid (Solvent B). Set the gradient: 10% B for 2.7 min, then to 20% B in 5.3 min, 28% B in 4 min, 35% B in another 18 min, to 40% B in 13 min, and 100% B in another 2 min. After holding 100% B for 5 min, lower the concentration to 10% B in 5 min and hold for the final 5 min.</li>
	</ol>
	</li>
	<li>Verify sample elution from the HPLC into the TIMS-TOF via nano-electrospray ionization (nESI) in positive ionization mode.</li>
</ol>
4. Data analysis
<ol>
	<li>Identify the peptide sequences and modification sites.
	<ol>
		<li>Prepare a theoretical list of peptides using ProteinProspector [https://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msdigest] under the MS-digest tool.
		<ol>
			<li>Perform a theoretical digest while taking into consideration the conditions of the digest (enzyme used), types of PTMs being searched for (e.g., mono-, di-, or trimethylation), the size range of peptides being searched for, as well as mass detection range and the potential number of missed cleavages.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Manually analyze the acquired data based on theoretical peptides (Figure 6)12.
	<ol>
		<li>Search for the masses at several charge states (+1 to +4) for each theoretical peptide are searched for.</li>
		<li>Following the initial identification of each m/z, select the peak and confirm the MS/MS using a theoretical list of fragmentation ions based on the peptide sequence, including PTMs. 
		NOTE: If the mobility of the identified peptide was known previously, this is also confirmed.</li>
	</ol>
	</li>
	<li>Calculate the relative abundances of various PTMs and report each modification as a percentage of the specified peptide sequence.
	<ol>
		<li>The relative abundance of each detected PTM is calculated using the following equation: 
		Relative abundance = Area of PTM/Total area of unmodified and PTMs for a given peptide</li>
	</ol>
	</li>
</ol>

Histone Proteomic Analysis and Post-Translational Modification Characterization

<ol>
	<li>Zhao, Z., 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=Epigenetic+modifications+of+histones+in+cancer.">Epigenetic modifications of histones in cancer.</a> Genome Biology. 20, 245 (2019).</li><li>Luger, K., Mader, A. W., Richmond, R. K., Sargen, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+&#197;+resolution.">Crystal structure of the nucleosome core particle at 2.8 &#197; resolution.</a> Nature. 389 (6648), 251-260 (1997).</li><li>Bentley, G. A., Lewit-Bentley, A., Finch, J. T., Podjarny, A. D., Roth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+16+&#197;+resolution.">Crystal structure of the nucleosome core particle at 16 &#197; resolution.</a> Journal of Molecular Biology. 176 (1), 55-75 (1984).</li><li>Kornberg, R. D., Thomas, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+structure:+Oligomers+of+the+histones:+The+histones+comprise+an+(F2A1)2(F3)2+tetramer,+a+different+oligomer+of+F2A2+and+F2B,+and+monomer+of+F1.">Chromatin structure: Oligomers of the histones: The histones comprise an (F2A1)2(F3)2 tetramer, a different oligomer of F2A2 and F2B, and monomer of F1.</a> Science. 184 (4139), 865-868 (1974).</li><li>Borghini, A., Cervelli, T., Galli, A., Andreassi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+modifications+in+atherosclerosis:+From+the+past+to+the+future.">DNA modifications in atherosclerosis: From the past to the future.</a> Atherosclerosis. 230 (2), 202-209 (2013).</li><li>Jeanne Dit Fouque, K., 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=34;Double-Down&amp;#34;+mass+spectrometry+of+histone+H4+proteoforms:+Tandem+ultraviolet-photon+and+mobility/mass-selected+electron+capture+dissociations.">34;Double-Down&amp;#34; mass spectrometry of histone H4 proteoforms: Tandem ultraviolet-photon and mobility/mass-selected electron capture dissociations.</a> Analytical Chemistry. 94 (44), 15377-15385 (2022).</li><li>Lawrence, M., Daujat, S., Schneider, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+thinking:+How+histone+modifications+regulate+gene+expression.">Lateral thinking: How histone modifications regulate gene expression.</a> Trends in Genetics. 32 (1), 42-56 (2016).</li><li>Bannister, A. J., 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=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Research. 21 (3), 381-395 (2011).</li><li>Meier, 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=Parallel+accumulation-serial+fragmentation+(PASEF):+Multiplying+sequencing+speed+and+sensitivity+by+synchronized+scans+in+a+trapped+ion+mobility+device.">Parallel accumulation-serial fragmentation (PASEF): Multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device.</a> Journal of Proteome Research. 14 (12), 5378-5387 (2015).</li><li>Chernushevich, I. V., Loboda, A. V., Thomson, 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=An+introduction+to+quadrupole-time-of-flight+mass+spectrometry.">An introduction to quadrupole-time-of-flight mass spectrometry.</a> Journal of Mass Spectrometry. 36 (8), 849-865 (2001).</li><li>Andrews, G. L., Simons, B. L., Young, J. B., Hawridge, A. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+characteristics+of+a+new+hybrid+quadrupole+time-of-flight+tandem+mass+spectrometer+(TripleTOF+5600).">Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600).</a> Analytical Chemistry. 83 (13), 5442-5446 (2011).</li><li>Bhanu, N. V., Sidoli, S., Garcia, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Workflow+for+ultra-rapid+analysis+of+histone+posttranslational+modifications+with+direct-injection+mass+spectrometry.">A Workflow for ultra-rapid analysis of histone posttranslational modifications with direct-injection mass spectrometry.</a> Bio-Protocol. 10 (18), e3756 (2020).</li><li>Xue, 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=Discovery+of+serum+biomarkers+for+pancreatic+adenocarcinoma+using+proteomic+analysis.">Discovery of serum biomarkers for pancreatic adenocarcinoma using proteomic analysis.</a> British Journal of Cancer. 103 (3), 391-400 (2010).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversed-phase+chromatography+with+multiple+fraction+concatenation+strategy+for+proteome+profiling+of+human+MCF10S+cells.">Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10S cells.</a> Proteomics. 11 (10), 2019-2026 (2011).</li><li>Mertins, P., 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=Integrated+proteomic+analysis+of+posttranslational+modifications+by+serial+enrichment.">Integrated proteomic analysis of posttranslational modifications by serial enrichment.</a> Nature Methods. 10 (7), 634-637 (2012).</li><li>Meier, F., Park, M. A., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapped+ion+mobility+spectrometry+and+parallel+accumulation-serial+fragmentation+in+proteomics.">Trapped ion mobility spectrometry and parallel accumulation-serial fragmentation in proteomics.</a> Molecular &amp; Cellular Proteomics. 20, 100138 (2021).</li><li>Romson, J., Emmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+calibration+options+for+accurate+electrospray+ionization+mass+spectrometry.">Mass calibration options for accurate electrospray ionization mass spectrometry.</a> International Journal of Mass Spectrometry. 467, 11619 (2021).</li><li>Dupree, E. J., Jayathirtha, M., Yorkie, H., Mihasan, M., Petre, B. A., Darie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+bottom-up+proteomics:+The+good,+the+bad,+and+the+future+of+this+field.">A critical review of bottom-up proteomics: The good, the bad, and the future of this field.</a> Proteomes. 8 (3), 14 (2020).</li><li>Ho, C. 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=Bottom-up+structural+proteomics:+cryoEM+of+protein+complexes+enriched+from+the+cellular+milieu.">Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu.</a> Nature Methods. 17 (1), 79-85 (2020).</li><li>Eickbush, T., Moudrianakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+core+complex:+an+octamer+assembled+by+two+sets+of+protein-protein+interactions.">The histone core complex: an octamer assembled by two sets of protein-protein interactions.</a> Biochemistry. 17 (23), 4955-4964 (1978).</li><li>Sadakierska-Chudy, A., Filip, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+view+of+the+epigenetic+landscape.+Part+II:+Histone+posttranslational+modification,+nucleosome+level,+and+chromatin+regulation+by+ncRNAs.">A Comprehensive view of the epigenetic landscape. Part II: Histone posttranslational modification, nucleosome level, and chromatin regulation by ncRNAs.</a> Neurotoxicity Research. 27 (2), 172-197 (2015).</li><li>Tan, H. T., Low, J., Lim, S. G., Chung, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+autoantibodies+as+biomarkers+for+early+cancer+detection.">Serum autoantibodies as biomarkers for early cancer detection.</a> The FEBS Journal. 276 (23), 6880-6904 (2009).</li><li>Huang, R. X., Zhou, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+response+pathways+and+targets+for+radiotherapy+sensitization+in+cancer.">DNA damage response pathways and targets for radiotherapy sensitization in cancer.</a> Signal Transduction and Targeted Therapy. 5 (1), 60 (2020).</li><li>Dantuma, N. P., van Attikum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+regulation+of+posttranslational+modifications+in+the+DNA+damage+response.">Spatiotemporal regulation of posttranslational modifications in the DNA damage response.</a> The EMBO Journal. 35 (1), 6-23 (2016).</li><li>Tanner, S., Pevzner, P. A., Bafna, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unrestrictive+identification+of+posttranslational+modifications+through+peptide+mass+spectrometry.">Unrestrictive identification of posttranslational modifications through peptide mass spectrometry.</a> Nature Protocols. 1 (1), 67-72 (2006).</li><li>Dolan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC+column+problems+everywhere+1.">LC column problems everywhere 1.</a> LCGC North America. 28 (9), 500-504 (2015).</li><li>Brusniak, M. K., 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+current+bioinformatic+solutions+for+analyzing+LC-MS+data+acquired+by+selected+reaction+monitoring+technology.">An assessment of current bioinformatic solutions for analyzing LC-MS data acquired by selected reaction monitoring technology.</a> Proteomics. 12 (8), 1176-1184 (2012).</li><li>Cham, J. A., Bianco, L., Bessant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+computational+resources+for+designing+selected+reaction+monitoring+transitions.">Free computational resources for designing selected reaction monitoring transitions.</a> Proteomics. 10 (6), 1106-1126 (2010).</li><li>Colangelo, C. M., Chung, L., Bruce, C., Cheung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+software+tools+for+design+and+analysis+of+large+scale+MRM+proteomic+datasets.">Review of software tools for design and analysis of large scale MRM proteomic datasets.</a> Methods. 61, 287-298 (2013).</li></ol>

Melvin A. Park and Matthew Willetts are employees of Bruker Daltonics Inc., the manufacturer of the timsTOF instrument.

Histones are basic proteins that regulate chromatin structure by interacting with DNA in the form of octamers consisting of the four core histones (two each of H2A, H2B, H3, and H4)20. Histones contain numerous lysine and arginine residues, which are readily modified, leading to extensive PTMs that alter the chromatin chemistry by influencing histone function or by binding to other cellular proteins21. PTMs can elicit biological responses by working in tandem, with specific...

In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)1,2,3,4. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation5. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair6. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA3,7. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins3,8.
A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process9. Time-of-flight (ToF) instruments acquire spectra at very high frequency9,10 (up to tens of kHz)11; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)9 and making them ideal for biological sample analysis10. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate9. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole9. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity9.
The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>-80 &#176;C Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td>Animal Origin-Free</td>
		</tr>
		<tr>
			<td>1 mL Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060710</td>
			<td>Finntip Flex 1000 &#956;L, nonsterile, nonfiltered, racked tips</td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-282-300</td>
			<td>Use these tubes for the simple and safe processing of sample volumes up to 1.5 mL</td>
		</tr>
		<tr>
			<td>10 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060100</td>
			<td>Finntip Flex, 10 &#956;L, nonsterile, non-filtered, racked</td>
		</tr>
		<tr>
			<td>10% NP-40</td>
			<td>Thermo Fisher Scientific</td>
			<td>28324</td>
			<td>NP-40 Surfact-Amps Detergent Solution</td>
		</tr>
		<tr>
			<td>10x Dulbecco&#8217;s PBS without Ca2+/Mg2+</td>
			<td>(Mediatech)</td>
			<td>MT21031CM</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Corning</td>
			<td>352196</td>
			<td>Falcon Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>200 &#181;L Gel-Loading Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-707-138</td>
			<td>Fisherbrand&#160;Gel-Loading Tips, 1&#8211;200 &#956;L</td>
		</tr>
		<tr>
			<td>200 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060310</td>
			<td>Finntip Flex 200&#956;L, nonsterile, nonfiltered, racked tips</td>
		</tr>
		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610737</td>
			<td>Premixed protein sample buffer for SDS-PAGE</td>
		</tr>
		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td>Falcon Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, V-Bottom 600 &#956;L</td>
			<td>Axygen</td>
			<td>P-DW-500-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>179124</td>
			<td>ACS reagent, &#8805;99.5%</td>
		</tr>
		<tr>
			<td>Acetonitrile (ACN)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A998</td>
			<td>HPLC, Fisher Chemical</td>
		</tr>
		<tr>
			<td>Acetonitrile with 0.1% Formic acid (v/v), LC/MS Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>LS120</td>
			<td>Optima LC/MS Grade, Thermo Scientific</td>
		</tr>
		<tr>
			<td>AEBSF</td>
			<td>Thermo Fisher Scientific</td>
			<td>328110500</td>
			<td>AEBSF hydrochloride, 98%</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate, NH4HCO3</td>
			<td>Sigma Aldrich</td>
			<td>09830</td>
			<td>BioUltra, &#8805;99.5% (T)</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution, NH4OH</td>
			<td>Sigma Aldrich</td>
			<td>AX1303</td>
			<td>Meets ACS Specifications, Meets Reagent Specifications for testing USP/NF monographs GR ACS</td>
		</tr>
		<tr>
			<td>Argon (Ar)</td>
			<td>Airgas</td>
			<td>AR HP 300</td>
			<td></td>
		</tr>
		<tr>
			<td>BEH C18 HPLC column</td>
			<td>Waters</td>
			<td>186003625</td>
			<td>XBridge Peptide BEH C18 Column, 300 &#197;, 5 &#181;m, 4.6 mm X 250 mm, 1K&#8211;15K</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Heat shock fraction, pH 7, &#8805;98%</td>
		</tr>
		<tr>
			<td>Calcium chloride, CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>Anhydrous, powder, &#8805;97%</td>
		</tr>
		<tr>
			<td>Cell dissociation buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>13151014</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic scoring wafer</td>
			<td>Restek</td>
			<td>20116</td>
			<td></td>
		</tr>
		<tr>
			<td>Compass DataAnalysis 6.0</td>
			<td>Bruker Datonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compass HyStar 6.2</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compass IsotopePattern</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compass timsControl 4.1</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad</td>
			<td>1610436</td>
			<td></td>
		</tr>
		<tr>
			<td>Deep Well, 96-Well Microplate, 2.0 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>89237526</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Cell Lifters</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;08100240</td>
			<td>Fisherbrand Cell Lifters; Disposable lifters quickly remove cell layers</td>
		</tr>
		<tr>
			<td>Disposable Pellet Pestles</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-141-363</td>
			<td>Fisherbrand&#160;Pellet Pestles; Resuspend protein and DNA pellets or grind soft tissue in microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Thermo Fisher Scientific</td>
			<td>P2325</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>Sigma Aldrich</td>
			<td>695076</td>
			<td>ACS reagent, &#8805;96%</td>
		</tr>
		<tr>
			<td>Fused silica capillary 75 &#956;m ID x 363 &#956;m OD</td>
			<td>(Molex (Polymicro)</td>
			<td>TSP075375</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>A38S</td>
			<td>Acetic Acid, Glacial (Certified ACS), Fisher Chemical</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipettes</td>
			<td>Sigma Aldrich</td>
			<td>BR747725-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Performance Liquid Chromatograph</td>
			<td>&#160;Shimadzu</td>
			<td></td>
			<td>Shimadzu Prominence 20 HPLC UFLC System</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, HCl</td>
			<td>Sigma Aldrich</td>
			<td>258148</td>
			<td>ACS reagent, 37%</td>
		</tr>
		<tr>
			<td>Hypercarb 30-40 &#956;m Carbon 150&#8211;300 &#197;</td>
			<td>Thermo Fisher Scientific</td>
			<td>60106-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypersep cartridge</td>
			<td>Thermo Fisher Scientific</td>
			<td>60109-404</td>
			<td></td>
		</tr>
		<tr>
			<td>LC/MS Calibration Standard, for ESI-ToF</td>
			<td>Agilent</td>
			<td>G1969-85000</td>
			<td>TuningMix</td>
		</tr>
		<tr>
			<td>Magnesium chloride, MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td>Anhydrous, &#8805;98%</td>
		</tr>
		<tr>
			<td>Methanol, for HPLC</td>
			<td>Thermo Fisher Scientific</td>
			<td>A454</td>
			<td>Optima for HPLC, Fisher Chemical&#160;&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tube Adapters</td>
			<td>GL Sciences</td>
			<td>501021514</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcystin</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-200-8727</td>
			<td>Enzo Life Sciences&#160;Microcystin-LA</td>
		</tr>
		<tr>
			<td>MS sample vial, LaPhaPack, Snap, 12 mm x 32 mm</td>
			<td>LEAP PAL Parts</td>
			<td>LAP.11190933</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>model: ND3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen (N2)</td>
			<td>Airgas</td>
			<td>NI UHP300</td>
			<td></td>
		</tr>
		<tr>
			<td>PEAKS Studio X+</td>
			<td>Bioinformatic Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator strips, Instachek</td>
			<td>Micro Essential Lab</td>
			<td>JR-113</td>
			<td>Model: Hydrion</td>
		</tr>
		<tr>
			<td>Potassium chloride, KCl</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td>ACS reagent, 99.0%&#8211;100.5%</td>
		</tr>
		<tr>
			<td>Pressure Injection Cell</td>
			<td>Next Advance</td>
			<td>&#160;model: PC77</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic Anhydride</td>
			<td>Sigma Aldrich</td>
			<td>8.00608</td>
			<td>For synthesis</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge (700&#8211;18,000 x g)</td>
			<td>NuAire, model: Nuwind</td>
			<td>NU-C200V</td>
			<td></td>
		</tr>
		<tr>
			<td>Reprosil-Pur 120 C18-AQ 3 &#956;m, 3 g</td>
			<td>ESI Source Solutions</td>
			<td>r13.aq.0003</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE Gels</td>
			<td>Bio-Rad</td>
			<td>4569035</td>
			<td>Any kD precast polyacrylamide gel, 8.6 cm &#215; 6.7 cm (W &#215; L), for use with Mini-PROTEAN Electrophoresis Cells</td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11079.06</td>
			<td>98+%</td>
		</tr>
		<tr>
			<td>Sodium chloride, NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S9888</td>
			<td>ACS reagent, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>SPE disk, C18</td>
			<td>VWR</td>
			<td>76333-134</td>
			<td>Empore SPE disk, C18, CDS Analytical, 90 mm x 0.5 mm, 12 &#181;m</td>
		</tr>
		<tr>
			<td>SpeedVac+ vacuum pump and plate rotor</td>
			<td>Savant</td>
			<td>model: SC210A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Millipore</td>
			<td>1.07651</td>
			<td>suitable for microbiology</td>
		</tr>
		<tr>
			<td>Sulfuric acid, H2SO4&#160;</td>
			<td>Sigma Aldrich</td>
			<td>339741</td>
			<td>99.999%</td>
		</tr>
		<tr>
			<td>TIMS-ToF Mass Spectrometer</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>model Tims tof ms</td>
		</tr>
		<tr>
			<td>Trichloroacetic acid solution, TCA</td>
			<td>Sigma Aldrich</td>
			<td>T0699</td>
			<td>6.1&#160;N</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma Aldrich</td>
			<td>302031</td>
			<td>Suitable for HPLC, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Triversa Nanomate</td>
			<td>Advion</td>
			<td>model: TR263</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypsinProtease, MS Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>90057</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>Thermo Fisher Scientific</td>
			<td>88881001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>88880017</td>
			<td></td>
		</tr>
		<tr>
			<td>Water with 0.1% Formic acid (v/v), LC/MS Grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>LS118</td>
			<td>Optima LC/MS Grade, Thermo Scientific&#160;</td>
		</tr>
	</tbody>
</table>

histone modification screening using liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry

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

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

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

A method is proposed using histone extraction protocol with 95%efficiency. Together with LC-TIMS-ToF MS/MS, in a short time, it is possible to obtain a screening for those PTMs present in the histone. And above all, the possibility of separating isomers.The separation of isomeric peptides though possible using tandem mass spectrometry alone is often difficult. By incorporating TIMS, we introduce an extra dimension of separation, simplifying many positional isomer identifications. In addition, passive technology increases the sensitivity of these experiments, further improving our ability to analyze epigenetic variations.With the increased ability to identify PTM positions, there comes the ability to further determine the correlation between various environmental or biological factors, and their effects on the epigenome. Methods that improve and facilitate analysis of histones can be applied to several research areas, including medical and environmental chemistry. The dynamics of protein confirmation in native or damaged states will be established from the implementation of TIMS MS/MS with HDX.Through LC-TIMS-ToF MS/MS and Nano LC-TIMS-ToF MS/MS, the differences in PTM existing in various biological sample will be charted.

A bottom-up proteomic workflow (Figure 7) typically involves the following: extraction of the target protein(s) from a crude sample, followed by quantifying the concentration of the protein(s), and then fractionation, usually by gel electrophoresis or liquid chromatography. After fractionation, the proteins are digested using a proteolytic enzyme (often trypsin), and finally, mass spectrometric analysis of the resulting peptides and protein identification using an established database<sup cl...

Watch this Scientific Journal Video about Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry at JoVE.com

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

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

histone-modification-screening-using-liquid-chromatography-trapped

Research

JoVE Journal

Chemistry

767 Views.  Florida International University. A method is proposed using histone extraction protocol with 95%efficiency. Together with LC-TIMS-ToF MS/MS, in a short time, it is possible to obtain a screening for those PTMs present in the histone. And above all, the possibility of separating isomers.The separation of isomeric peptides though possible using tandem mass spectrometry alone is often difficult. By incorporating TIMS, we introduce an extra dimension of separation, simplifying many positional isomer identifications. In ad...