Name: Forfatter Spotlight: Forbedret histon PTM-isomeridentifikasjon gjennom 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 materialet er basert på arbeid støttet av National Science Foundation under Grant No. HRD-1547798 og tilskudd nr. HRD-2111661. Disse NSF-tilskuddene ble tildelt Florida International University som en del av Centers of Research Excellence in Science and Technology (CREST) -programmet. Dette er bidragsnummer 1672 fra Institute of Environment, et fremtredende program ved Florida International University. Ytterligere støtte ble gitt av National Institute of Health under tilskudd nr. R21AI135469 til Francisco Fernandez-Lima og Grant nr. R01HD106051 til Benjamin A. Garcia, samt av National Science Foundation under Grant nr. CHE-2127882 til Benjamin A. Garcia. Forfatterne ønsker å anerkjenne den første støtten fra Dr. Mario Gomez Hernandez under innledende metodeutvikling.

MERK: Histonprøver ble ekstrahert ved hjelp av en metode tilpasset fra Bhanu et al. (2020) 12.
1. Forberedelse av prøver
<ol><li>Høsting av dyrkede celler<ol><li>Når cellene er 80 % sammenflytende, må du sørge for at de er levedyktige ved hjelp av trypanblå ekskludering. MERK: En HeLa S3-cellelinje ble brukt til disse eksperimentene, men denne metoden kan brukes på alle dyrkede celler.</li>
		<li>Aspirer mediet, og påfør deretter 5 ml 1x ...

Histonproteomisk analyse og karakterisering av post-translasjonell modifikasjon

<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 grunnleggende proteiner som regulerer kromatinstrukturen ved å interagere med DNA i form av oktamere som består av de fire kjernehistonene (to hver av H2A, H2B, H3 og H4)20. Histoner inneholder mange lysin- og argininrester, som lett modifiseres, noe som fører til omfattende PTM som endrer kromatinkjemien ved å påvirke histonfunksjonen eller ved binding til andre cellulære proteiner21. PTM kan fremkalle biologiske responser ved å jobbe sammen, med spesifi...

I eukaryote celler pakkes DNA som kromatin i funksjonelle enheter kalt nukleosomer. Disse enhetene består av en oktamer med fire kjernehistoner (to hver av H2A, H2B, H3 og H4)1,2,3,4. Histoner er blant de mest tallrike og svært konserverte proteinene i eukaryoter, som i stor grad er ansvarlige for genregulering5. Histon posttranslasjonelle modifikasjoner (PTM) spiller en stor rolle i reguleringen av kromatindynamikk og rigger ulike biologiske prosesser som DNA-transkripsjon, replikasjon og reparasjon6. PTM forekommer primært på den tilgjengelige overflaten av de N-terminale områdene av histoner som er i kontakt med DNA 3,7. Imidlertid påvirker hale- og kjernemodifikasjoner kromatinstrukturen, endrer internukleosominteraksjoner og rekrutterer spesifikke proteiner 3,8.
En aktuell utfordring under væskekromatografi-massespektrometri (LC-MS)-basert proteomikk er potensiell co-eluering av analytter av interesse. Når det gjelder dataavhengige analyser (DDA), betyr dette potensielt tap av flere forløperioner under MS/MS-innsamlingsprosessen9. ToF-instrumenter (time-of-flight) oppnår spektra med svært høy frekvens 9,10 (opptil titalls kHz)11; Dette gjør dem i stand til raskt å skanne de totale forløperionene i en kompleks prøve (MS1), og dermed love optimal følsomhet og MS / MS-sekvenseringshastigheter (opptil 100 Hz) 9 og gjør dem ideelle for biologisk prøveanalyse10. Likevel er følsomheten som er tilgjengelig ved disse høye skannehastighetene begrenset av MS / MS-hastigheten9. Tilsetningen av fanget ionmobilitetsspektrometri (TIMS) i kombinasjon med et ortogonalt kvadrupol time-of-flight (qToF) massespektrometer ble brukt til å redusere disse begrensningene. I TIMS akkumuleres alle forløperioner i tandem og elueres som en funksjon av deres mobilitet, i stedet for å velge enkeltforløpermasser med en kvadrupol9. Parallell akkumulering-seriell fragmentering (PASEF) tillater hundrevis av MS/MS-hendelser per sekund uten tap av følsomhet9.
Hovedmålet med dette arbeidet var å vise den siste utviklingen av DDA ved hjelp av parallell akkumulering i mobilitetsfellen etterfulgt av sekvensiell fragmentering og kollisjonsindusert dissosiasjon (CID). Histon-PTM-er ble trygt tildelt basert på deres oppbevaringstider (RT), mobiliteter og fragmenteringsmønstre.

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			<td>Thermo Fisher Scientific</td>
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			<td>Ceramic scoring wafer</td>
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			<td>Compass DataAnalysis 6.0</td>
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			<td>Compass HyStar 6.2</td>
			<td>Bruker Daltonics</td>
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			<td>Compass IsotopePattern</td>
			<td>Bruker Daltonics</td>
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			<td>Compass timsControl 4.1</td>
			<td>Bruker Daltonics</td>
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			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad</td>
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			<td>Deep Well, 96-Well Microplate, 2.0 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>89237526</td>
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		</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 svært rikelig og konservert blant eukaryoter og spiller en stor rolle i genregulering som følge av strukturer kjent som posttranslasjonelle modifikasjoner (PTM). Identifisering av posisjonen og naturen til hver PTM eller mønster av PTM i referanse til eksterne eller genetiske faktorer gjør at denne informasjonen kan statistisk korreleres med biologiske responser som DNA-transkripsjon, replikasjon eller reparasjon. I det foreliggende arbeidet beskrives en analytisk protokoll med høy gjennomstrømning for deteksjon av histon PTM fra biologiske prøver. Bruken av komplementær væskekromatografi, mobilitetsspektrometri for fangede ioner og massespektrometri (LC-TIMS-ToF MS/MS) muliggjør separasjon og PTM-tildeling av de mest biologisk relevante modifikasjonene i en enkelt analyse. Den beskrevne tilnærmingen utnytter den siste utviklingen i avhengig datainnsamling (DDA) ved bruk av parallell akkumulering i mobilitetsfellen, etterfulgt av sekvensiell fragmentering og kollisjonsindusert dissosiasjon. Histon-PTM-er tildeles trygt basert på deres oppbevaringstid, 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-isomeridentifikasjon gjennom LC-TIMS-ToF MS/MS og PASEF

Histone Modification Screening ved bruk av væskekromatografi, Trapped Ion Mobility Spectrometry og 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.

En bottom-up proteomisk arbeidsflyt (figur 7) innebærer vanligvis følgende: ekstraksjon av målproteinet (e) fra en råprøve, etterfulgt av kvantifisering av konsentrasjonen av proteinet (e), og deretter fraksjonering, vanligvis ved gelelektroforese eller væskekromatografi. Etter fraksjonering fordøyes proteinene ved hjelp av et proteolytisk enzym (ofte trypsin), og til slutt massespektrometrisk analyse av de resulterende peptidene og proteinidentifikasjon ved hjelp av en etablert datab...

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 arbeidsflyt basert på væskekromatografi, mobilitetsspektrometri for fangede ioner og time-of-flight massespektrometri (LC-TIMS-ToF MS/MS) for høy konfidensialitet og svært reproduserbar "bottom-up"-analyse av histonmodifikasjoner og identifikasjon basert på hovedparametere (retensjonstid [RT], kollisjonstverrsnitt [CCS] og nøyaktig 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 ...

En metode foreslås ved bruk av histonekstraksjonsprotokoll med 95% effektivitet. Sammen med LC-TIMS-ToF MS/MS er det på kort tid mulig å få en screening for de PTM-ene som er tilstede i histonen. Og fremfor alt, muligheten for å separere isomerer.Separasjonen av isomere peptider, selv om det er mulig ved bruk av tandemmassespektrometri alene, er ofte vanskelig. Ved å inkorporere TIMS introduserer vi en ekstra separasjonsdimensjon, noe som forenkler mange posisjonelle isomeridentifikasjoner. I tillegg øker passiv teknologi følsomheten til disse eksperimentene, noe som ytterligere forbedrer vår evne til å analysere epigenetiske variasjoner.Med økt evne til å identifisere PTM-posisjoner, kommer det muligheten til ytterligere å bestemme sammenhengen mellom ulike miljømessige eller biologiske faktorer, og deres effekter på epigenomet. Metoder som forbedrer og forenkler analyse av histoner kan brukes på flere forskningsområder, inkludert medisinsk og miljøkjemi. Dynamikken i proteinbekreftelse i native eller skadede tilstander vil bli etablert fra implementeringen av TIMS MS / MS med HDX.Gjennom LC-TIMS-ToF MS/MS og Nano LC-TIMS-ToF MS/MS vil forskjellene i PTM som finnes i ulike biologiske prøver bli kartlagt.

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).