Name: Forfatter Spotlight: Forbedret histon PTM-isomeridentifikation gennem LC-TIMS-ToF MS/MS og PASEF
Uploaded: 2024-01-12T12:50:00
Description: 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

Dette materiale er baseret på arbejde støttet af National Science Foundation under bevilling nr. HRD-1547798 og bevillingsnr. HRD-2111661. Disse NSF-tilskud blev tildelt Florida International University som en del af Centers of Research Excellence in Science and Technology (CREST) -programmet. Dette er bidrag nummer 1672 fra Institute of Environment, et fremtrædende program ved Florida International University. Yderligere støtte blev ydet af National Institute of Health under tilskud nr. R21AI135469 til Francisco Fernandez-Lima og Grant No. R01HD106051 til Benjamin A. Garcia, samt af National Science Foundation under bevilling nr. CHE-2127882 til Benjamin A. Garcia. Forfatterne vil gerne anerkende den indledende støtte fra Dr. Mario Gomez Hernandez under den indledende metodeudvikling.

BEMÆRK: Histonprøver blev ekstraheret ved hjælp af en metode tilpasset fra Bhanu et al. (2020)12.
1. Forberedelse af prøver
<ol><li>Høstning af dyrkede celler<ol><li>Når celler er 80% sammenflydende, skal du sikre dig, at de er levedygtige ved hjælp af trypanblå udelukkelse. BEMÆRK: En HeLa S3-cellelinje blev brugt til disse eksperimenter, men denne metode kan anvendes på alle dyrkede celler.</li>
		<li>Opsug mediet, og påfør derefter 5...

Histonproteomisk analyse og karakterisering af post-translationel modifikation

<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>

Histoner er basiske proteiner, der regulerer kromatinstrukturen ved at interagere med DNA i form af oktamerer bestående af de fire kernehistoner (to hver af H2A, H2B, H3 og H4)20. Histoner indeholder talrige lysin- og argininrester, som let modificeres, hvilket fører til omfattende PTM'er, der ændrer kromatinkemien ved at påvirke histonfunktionen eller ved binding til andre cellulære proteiner21. PTM'er kan fremkalde biologiske reaktioner ved at arbejde sammen, idet sp...

I eukaryote celler pakkes DNA som kromatin i funktionelle enheder kaldet nukleosomer. Disse enheder består af en oktam af fire kernehistoner (to hver af H2A, H2B, H3 og H4)1,2,3,4. Histoner er blandt de mest rigelige og højt konserverede proteiner i eukaryoter, som i høj grad er ansvarlige for genregulering5. Histone posttranslationelle modifikationer (PTM'er) spiller en stor rolle i reguleringen af kromatindynamik og rigger forskellige biologiske processer såsom DNA-transkription, replikation og reparation6. PTM'er forekommer primært på den tilgængelige overflade af de N-terminale regioner af histoner, der er i kontakt med DNA 3,7. Imidlertid påvirker hale- og kernemodifikationer kromatinstrukturen, ændrer internukleosominteraktioner og rekrutterer specifikke proteiner 3,8.
En aktuel udfordring under proteomics baseret på væskekromatografi-massespektrometri (LC-MS) er den potentielle sameluering af analysander af interesse. I tilfælde af dataafhængige analyser (DDA) betyder dette det potentielle tab af flere prækursorioner under MS/MS-erhvervelsesprocessen9. Time-of-flight-instrumenter (ToF) får spektre ved meget høj frekvens 9,10 (op til snesevis af kHz)11 dette gør dem i stand til hurtigt at scanne de samlede prækursorioner i en kompleks prøve (MS1), hvilket lover optimal følsomhed og MS/MS-sekventeringshastigheder (op til 100 Hz)9 og gør dem ideelle til biologisk prøveanalyse10. Ikke desto mindre er den følsomhed, der er tilgængelig ved disse høje scanningshastigheder, begrænset af MS / MS-hastigheden9. Tilføjelsen af fanget ionmobilitetsspektrometri (TIMS) i kombination med et ortogonalt quadrupole time-of-flight (qToF) massespektrometer blev brugt til at afbøde disse begrænsninger. I TIMS akkumuleres alle forløbere i tandem og elueres som en funktion af deres mobilitet snarere end at vælge enkeltforløbermasser med en quadrupol9. Parallel akkumuleringsseriel fragmentering (PASEF) giver mulighed for hundredvis af MS/MS-hændelser pr. sekund uden tab af følsomhed9.
Hovedformålet med dette arbejde var at vise den seneste udvikling i Dohaudviklingsdagsordenen ved hjælp af parallel akkumulering i mobilitetsfælden efterfulgt af sekventiel fragmentering og kollisionsinduceret dissociation (CID). Histone PTM'er blev med sikkerhed tildelt baseret på deres retentionstider (RT'er), mobilitet og fragmenteringsmønstre.

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			<td>-80 &#176;C Freezer</td>
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			<td>1x Phosphate Buffered Saline (PBS), pH 7.4</td>
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			<td>352196</td>
			<td>Falcon Conical Centrifuge Tubes</td>
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			<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>
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			<td>200 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060310</td>
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			<td>96-well flat bottom plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565501</td>
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			<td>96-well plate, V-Bottom 600 &#956;L</td>
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			<td>Thermo Fisher Scientific</td>
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			<td>AEBSF</td>
			<td>Thermo Fisher Scientific</td>
			<td>328110500</td>
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			<td>Ammonium bicarbonate, NH4HCO3</td>
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			<td>09830</td>
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			<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>
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			<td>AR HP 300</td>
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			<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>
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			<td>Bovine Serum Albumin (BSA)</td>
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			<td>A7906</td>
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			<td>C4901</td>
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			<td>Thermo Fisher Scientific</td>
			<td>13151014</td>
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			<td>Ceramic scoring wafer</td>
			<td>Restek</td>
			<td>20116</td>
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			<td>Compass DataAnalysis 6.0</td>
			<td>Bruker Datonics</td>
			<td></td>
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			<td>Compass HyStar 6.2</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Compass IsotopePattern</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Compass timsControl 4.1</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad</td>
			<td>1610436</td>
			<td></td>
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			<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

Histonproteiner er meget rigelige og konserverede blandt eukaryoter og spiller en stor rolle i genregulering som følge af strukturer kendt som posttranslationelle modifikationer (PTM'er). Identifikation af positionen og arten af hver PTM eller mønster af PTM'er i forhold til eksterne eller genetiske faktorer gør det muligt at korrelere disse oplysninger statistisk med biologiske reaktioner såsom DNA-transkription, replikation eller reparation. I dette arbejde beskrives en analytisk protokol med høj kapacitet til påvisning af histon-PTM'er fra biologiske prøver. Anvendelsen af komplementær væskekromatografi, fanget ionmobilitetsspektrometri og time-of-flight massespektrometri (LC-TIMS-ToF MS/MS) muliggør separation og PTM-tildeling af de mest biologisk relevante modifikationer i en enkelt analyse. Den beskrevne tilgang udnytter den seneste udvikling inden for afhængig dataindsamling (DDA) ved hjælp af parallel akkumulering i mobilitetsfælden efterfulgt af sekventiel fragmentering og kollisionsinduceret dissociation. Histone PTM'er tildeles trygt baseret på deres retentionstid, mobilitet og fragmenteringsmønster.

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.

Forfatter Spotlight: Forbedret histon PTM-isomeridentifikation gennem LC-TIMS-ToF MS/MS og PASEF

Histonmodifikationsscreening ved hjælp af væskekromatografi, fanget ionmobilitetsspektrometri og time-of-flight-massespektrometri

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.

En bottom-up proteomisk arbejdsgang (figur 7) involverer typisk følgende: ekstraktion af målproteinet (-erne) fra en rå prøve efterfulgt af kvantificering af koncentrationen af proteinet (proteinerne) og derefter fraktionering, normalt ved gelelektroforese eller væskekromatografi. Efter fraktionering fordøjes proteinerne under anvendelse af et proteolytisk enzym (ofte trypsin) og endelig massespektrometrisk analyse af de resulterende peptider og proteinidentifikation under anvendelse a...

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

En analytisk arbejdsgang baseret på væskekromatografi, fanget ionmobilitetsspektrometri og time-of-flight massespektrometri (LC-TIMS-ToF MS / MS) for høj tillid og meget reproducerbar "bottom-up" analyse af histonmodifikationer og identifikation baseret på hovedparametre (retentionstid [RT], kollisionstværsnit [CCS] og nøjagtigt masse-til-ladning [m / z] forhold).

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

Der foreslås en metode ved anvendelse af histonekstraktionsprotokol med 95%effektivitet. Sammen med LC-TIMS-ToF MS / MS er det på kort tid muligt at få en screening for de PTM'er, der er til stede i histonen. Og frem for alt muligheden for at adskille isomerer.Adskillelsen af isomere peptider, selvom det er muligt ved anvendelse af tandemmassespektrometri alene, er ofte vanskelig. Ved at inkorporere TIMS introducerer vi en ekstra dimension af adskillelse, hvilket forenkler mange positionelle isomeridentifikationer. Derudover øger passiv teknologi følsomheden af disse eksperimenter, hvilket yderligere forbedrer vores evne til at analysere epigenetiske variationer.Med den øgede evne til at identificere PTM-positioner kommer der evnen til yderligere at bestemme sammenhængen mellem forskellige miljømæssige eller biologiske faktorer og deres virkninger på epigenomet. Metoder, der forbedrer og letter analyse af histoner, kan anvendes på flere forskningsområder, herunder medicinsk og miljøkemi. Dynamikken i proteinbekræftelse i indfødte eller beskadigede tilstande vil blive etableret fra implementeringen af TIMS MS / MS med HDX.Gennem LC-TIMS-ToF MS / MS og Nano LC-TIMS-ToF MS / MS vil forskellene i PTM, der findes i forskellige biologiske prøver, blive kortlagt.

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

Research

JoVE Journal

Chemistry

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.

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).