Name: Autor em destaque: Identificação aprimorada de isômeros PTM de histonas por meio de LC-TIMS-ToF MS / MS e 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

Este material é baseado em trabalho apoiado pela National Science Foundation sob o Grant No. HRD-1547798 e Bolsa nº. HRD-2111661. Essas bolsas da NSF foram concedidas à Florida International University como parte do Programa Centers of Research Excellence in Science and Technology (CREST). Esta é a contribuição número 1672 do Institute of Environment, um programa proeminente da Florida International University. O apoio adicional foi fornecido pelo Instituto Nacional de Saúde sob a Bolsa nº. R21AI135469 a Francisco Fernandez-Lima e Grant nº. R01HD106051 a Benjamin A. Garcia, bem como pela National Science Foundation sob o Grant No. CHE-2127882 para Benjamin A. Garcia. Os autores gostariam de agradecer o apoio inicial do Dr. Mario Gomez Hernandez durante o desenvolvimento inicial do método.

NOTA: As amostras de histona foram extraídas usando um método adaptado de Bhanu et al (2020)12.
1. Preparação da amostra
<ol><li>Colheita de células cultivadas<ol><li>Quando as células são 80% confluentes, certifique-se de que são viáveis usando a exclusão de azul de tripano. NOTA: Uma linhagem celular HeLa S3 foi usada para esses experimentos, mas este método pode ser aplicado a qualquer célula cultivada.</li>
		<li>Aspirar o meio e, e...

Análise proteômica de histonas e caracterização de modificação pós-tradução

<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>

As histonas são proteínas básicas que regulam a estrutura da cromatina interagindo com o DNA na forma de octâmeros constituídos pelas quatro histonas centrais (duas de H2A, H2B, H3 e H4)20. As histonas contêm numerosos resíduos de lisina e arginina, que são prontamente modificados, levando a extensas MPTs que alteram a química da cromatina influenciando a função das histonas ou ligando-se a outras proteínas celulares21. As MPT podem provocar respostas biológica...

Em células eucarióticas, o DNA é empacotado como cromatina em unidades funcionais chamadas nucleossomos. Essas unidades são compostas por um octâmero de quatro histonas de núcleo (duas de H2A, H2B, H3 e H4)1,2,3,4. As histonas estão entre as proteínas mais abundantes e altamente conservadas em eucariotos, as quais são as grandes responsáveis pela regulação gênica5. As modificações pós-traducionais de histona (MPTs) desempenham um grande papel na regulação da dinâmica da cromatina e rigger vários processos biológicos, como transcrição, replicação e reparo do DNA6. As MPT ocorrem principalmente na superfície acessível das regiões N-terminais das histonas que estão em contato com o DNA 3,7. Entretanto, modificações na cauda e no núcleo influenciam a estrutura da cromatina, alterando as interações internucleossomas e recrutando proteínas específicas 3,8.
Um desafio atual durante a proteômica baseada em cromatografia líquida-espectrometria de massas (LC-MS) é a potencial co-eluição de analitos de interesse. No caso das análises dependentes de dados (DDA), isso se traduz na perda potencial de vários íons precursores durante o processo de aquisição de MS/MS9. Os instrumentos Time-of-flight (ToF) adquirem espectros em frequência muito alta 9,10 (até dezenas de kHz)11; isso os torna capazes de escanear rapidamente os íons precursores totais dentro de uma amostra complexa (MS1), prometendo sensibilidade ótima e taxas de sequenciamento MS/MS (até 100 Hz)9 e tornando-os ideais para análise de amostras biológicas10. No entanto, a sensibilidade disponível nessas altas taxas de exame é limitada pela taxa MS/MS9. A adição de espectrometria de mobilidade iônica aprisionada (TIMS) em combinação com um espectrômetro de massa ortogonal quadrupolo time-of-flight (qToF) foi usada para mitigar essas limitações. No TIMS, todos os íons precursores são acumulados em tandem e eluídos em função de sua mobilidade, em vez de selecionar massas precursoras únicas com um quadrupolo9. A acumulação paralela-fragmentação seriada (PASEF) permite centenas de eventos MS/MS por segundo sem qualquer perda de sensibilidade9.
O objetivo principal deste trabalho foi mostrar os desenvolvimentos recentes da DDA usando acumulação paralela na armadilha de mobilidade, seguida de fragmentação sequencial e dissociação induzida por colisão (DDIC). Os PTMs Histone foram atribuídos com confiança com base em seus tempos de retenção (RTs), mobilidades e padrões de fragmentação.

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			<td>-80 &#176;C Freezer</td>
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			<td>Thermo Fisher Scientific</td>
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			<td>Fisherbrand&#160;Gel-Loading Tips, 1&#8211;200 &#956;L</td>
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			<td>200 &#181;L Pipette Tips</td>
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			<td>12565501</td>
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			<td>96-well plate, V-Bottom 600 &#956;L</td>
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			<td>P-DW-500-C-S</td>
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			<td>179124</td>
			<td>ACS reagent, &#8805;99.5%</td>
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			<td>Thermo Fisher Scientific</td>
			<td>A998</td>
			<td>HPLC, Fisher Chemical</td>
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			<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>
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			<td>AEBSF</td>
			<td>Thermo Fisher Scientific</td>
			<td>328110500</td>
			<td>AEBSF hydrochloride, 98%</td>
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			<td>Ammonium bicarbonate, NH4HCO3</td>
			<td>Sigma Aldrich</td>
			<td>09830</td>
			<td>BioUltra, &#8805;99.5% (T)</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>Argon (Ar)</td>
			<td>Airgas</td>
			<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>
			<td>Heat shock fraction, pH 7, &#8805;98%</td>
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			<td>Calcium chloride, CaCl2</td>
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			<td>C4901</td>
			<td>Anhydrous, powder, &#8805;97%</td>
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			<td>Cell dissociation buffer</td>
			<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>
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			<td>Compass HyStar 6.2</td>
			<td>Bruker Daltonics</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>
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			<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

As proteínas histonas são altamente abundantes e conservadas entre os eucariotos e desempenham um grande papel na regulação gênica como resultado de estruturas conhecidas como modificações pós-traducionais (MPTs). Identificar a posição e a natureza de cada PTM ou padrão de PTMs em referência a fatores externos ou genéticos permite que essa informação seja estatisticamente correlacionada com respostas biológicas, como transcrição, replicação ou reparo de DNA. No presente trabalho, um protocolo analítico de alto rendimento para a detecção de PTMs de histonas a partir de amostras biológicas é descrito. O uso de cromatografia líquida complementar, espectrometria de mobilidade iônica aprisionada e espectrometria de massas por tempo de voo (LC-TIMS-ToF MS/MS) permite a separação e atribuição de PTM das modificações biologicamente mais relevantes em uma única análise. A abordagem descrita aproveita os recentes desenvolvimentos na aquisição de dados dependentes (DDA) usando acumulação paralela na armadilha de mobilidade, seguida por fragmentação sequencial e dissociação induzida por colisão. Os PTMs Histone são atribuídos com confiança com base em seu tempo de retenção, mobilidade e padrão de fragmentação.

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.

Autor em destaque: Identificação aprimorada de isômeros PTM de histonas por meio de LC-TIMS-ToF MS / MS e PASEF

Triagem de modificação de histona usando cromatografia líquida, espectrometria de mobilidade iônica aprisionada e espectrometria de massa por tempo de voo

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.

Um fluxo de trabalho proteômico ascendente (Figura 7) tipicamente envolve o seguinte: extração da(s) proteína(s) alvo(s) de uma amostra bruta, seguida da quantificação da concentração da(s) proteína(s) e, em seguida, fracionamento, geralmente por eletroforese em gel ou cromatografia líquida. Após o fracionamento, as proteínas são digeridas usando uma enzima proteolítica (muitas vezes tripsina) e, finalmente, análise espectrométrica de massa dos peptídeos resultantes e identi...

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

Um fluxo de trabalho analítico baseado em cromatografia líquida, espectrometria de mobilidade iônica aprisionada e espectrometria de massa por tempo de voo (LC-TIMS-ToF MS/MS) para alta confiança e análise "bottom-up" altamente reprodutível de modificações e identificação de histonas com base em parâmetros principais (tempo de retenção [TR], seção transversal de colisão [CCS] e relação massa-carga [m/z] precisa).

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

Um método é proposto usando protocolo de extração de histonas com 95% de eficiência. Juntamente com LC-TIMS-ToF MS/MS, em um curto espaço de tempo, é possível obter uma triagem para as MPTs presentes na histona. E, sobretudo, a possibilidade de separar isômeros.A separação de peptídeos isoméricos, embora possível usando espectrometria de massa em tandem sozinha, é muitas vezes difícil. Ao incorporar o TIMS, introduzimos uma dimensão extra de separação, simplificando muitas identificações de isômeros posicionais. Além disso, a tecnologia passiva aumenta a sensibilidade desses experimentos, melhorando ainda mais nossa capacidade de analisar variações epigenéticas.Com o aumento da capacidade de identificar posições de PTM, vem a capacidade de determinar ainda mais a correlação entre vários fatores ambientais ou biológicos, e seus efeitos sobre o epigenoma. Métodos que melhoram e facilitam a análise de histonas podem ser aplicados a diversas áreas de pesquisa, incluindo química médica e ambiental. A dinâmica de confirmação de proteínas em estados nativos ou lesados será estabelecida a partir da implementação do TIMS MS/MS com HDX.Através de LC-TIMS-ToF MS/MS e Nano LC-TIMS-ToF MS/MS, as diferenças de PTM existentes em várias amostras biológicas serão mapeadas.

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).