Name: 저자 스포트라이트: LC-TIMS-ToF MS/MS 및 PASEF를 통한 향상된 Histone PTM 이성질체 식별
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

이 자료는 미국 국립과학재단(National Science Foundation)의 보조금 번호(Grant No.) HRD-1547798 및 보조금 번호 HRD-2111661입니다. 이 NSF 보조금은 CREST(Centers of Research Excellence in Science and Technology) 프로그램의 일환으로 Florida International University에 수여되었습니다. 이것은 Florida International University의 탁월한 프로그램인 Institute of Environment의 기부 번호 1672입니다. 추가 지원은 미국 국립보건원(National Institute of Health)에서 보조금 번호로 제공되었습니다. 프란시스코 페르난데스-리마(Francisco Fernandez-Lima)와 그랜트 번호(Grant No.)R21AI135469 벤자민 A. 가르시아(Benjamin A. Garcia)와 국립과학재단(National Science Foundation)의 그랜트 번호(Grant No.)R01HD106051 CHE-2127882에서 Benjamin A. Garcia에게. 저자는 초기 분석법 개발 과정에서 Mario Gomez Hernandez박사의 초기 지원에 감사를 표하고자 합니다.

참고: 히스톤 샘플은 Bhanu et al. (2020)12에서 채택된 방법을 사용하여 추출되었습니다.
1. 샘플 준비
<ol><li>배양된 세포 채취<ol><li>세포가 80% 합류하면 트리판 블루 배제를 사용하여 세포가 생존 가능한지 확인합니다. 참고: 이 실험에는 HeLa S3 세포주가 사용되었지만, 이 방법은 모든 배양 세포에 적용할 수 있습니다.</li>
		<li>배지를 흡인한 다음 ...

Histone Proteomic Analysis 및 Post-Translational Modification Characterization (히스톤 단백질체 분석 및 번역 후 변형 특성 분석)

<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>

히스톤은 4개의 핵심 히스톤(H2A, H2B, H3 및 H4 각각 2개)으로 구성된 옥타머 형태의 DNA와 상호 작용하여 염색질 구조를 조절하는 염기성 단백질입니다.20. 히스톤에는 수많은 라이신 및 아르기닌 잔기가 포함되어 있으며, 이는 쉽게 변형되어 히스톤 기능에 영향을 미치거나 다른 세포 단백질에 결합하여 염색질 화학을 변화시키는 광범위한 PTM을 유발합니다21. PTM은...

진핵 세포에서 DNA는 뉴클레오솜(nucleosome)이라고 하는 기능적 단위로 염색질로 포장됩니다. 이 단위는 4 개의 코어 히스톤 (H2A, H2B, H3 및 H4 각각 2 개) 1,2,3,4의 옥타머로 구성됩니다. 히스톤은 진핵생물에서 가장 풍부하고 고도로 보존된 단백질 중 하나로, 유전자 조절에 크게 관여한다5. 히스톤 번역후 변형(PTM)은 염색질 역학의 조절에 큰 역할을 하며 DNA 전사, 복제 및 복구와 같은 다양한 생물학적 과정을 조작한다6. PTM은 주로 DNA 3,7과 접촉하는 히스톤의 N- 말단 영역의 접근 가능한 표면에서 발생합니다. 그러나 꼬리와 코어 변형은 염색질 구조에 영향을 미쳐 뉴클레오솜 간 상호 작용을 변경하고 특정 단백질을 모집합니다 3,8.
액체 크로마토그래피-질량분석법(LC-MS) 기반 단백질체학의 현재 과제는 관심 분석물질의 잠재적인 동시 용리입니다. 데이터 의존적 분석(DDA)의 경우, 이는 MS/MS 수집 공정 중 여러 전구체 이온의 잠재적 손실로 해석됩니다9. ToF(Time-of-Flight) 기기는 매우 높은 주파수 9,10(최대 수십 kHz)11에서 스펙트럼을 획득합니다. 따라서 복잡한 시료(MS1) 내의 전체 전구체 이온을 빠르게 스캔할 수 있으므로 최적의 감도와 MS/MS 염기서열분석 속도(최대 100Hz)9를 약속하고 생물학적 시료 분석에 이상적입니다10. 그럼에도 불구하고 이러한 높은 스캔 속도에서 사용할 수 있는 감도는 MS/MS 속도9에 의해 제한됩니다. 이러한 한계를 완화하기 위해 직교 사중극자 비행 시간(qToF) 질량 분석기와 함께 TIMS(Trapped Ion Mobility Spectrometry)를 추가했습니다. TIMS에서 모든 전구체 이온은 사중극자9로 단일 전구체 질량을 선택하는 대신 이동성의 함수로 나란히 축적되고 용리됩니다. PASEF(Parallel Accumulation-Serial Fragmentation)는 감도 손실 없이 초당 수백 개의 MS/MS 이벤트를 허용합니다9.
이 연구의 주요 목표는 이동성 트랩의 병렬 축적에 이어 순차적 단편화 및 충돌 유도 해리(CID)를 사용하여 DDA의 최근 발전을 보여주는 것이었습니다. Histone PTM은 머무름 시간(RT), 이동성 및 단편화 패턴을 기반으로 자신 있게 할당되었습니다.

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

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

히스톤 단백질은 진핵생물 사이에서 매우 풍부하고 보존되어 있으며 번역 후 변형(PTM)으로 알려진 구조의 결과로 유전자 조절에 큰 역할을 합니다. 외부 또는 유전적 요인과 관련하여 각 PTM의 위치 및 특성 또는 PTM 패턴을 식별하면 이 정보를 DNA 전사, 복제 또는 복구와 같은 생물학적 반응과 통계적으로 연관시킬 수 있습니다. 본 연구에서는 생물학적 샘플에서 히스톤 PTM을 검출하기 위한 고처리량 분석 프로토콜에 대해 설명합니다. 상보적 액체 크로마토그래피, 포획 이온 이동성 분광법 및 ToF(Time-of-Flight) 질량분석법(LC-TIMS-ToF MS/MS)을 사용하면 단일 분석에서 생물학적으로 가장 관련성이 높은 변형을 분리하고 PTM을 할당할 수 있습니다. 설명된 접근 방식은 이동성 트랩에서 병렬 축적을 사용한 후 순차적 단편화 및 충돌 유도 해리를 사용하는 종속 데이터 수집(DDA)의 최근 개발을 활용합니다. Histone PTM은 머무름 시간, 이동성 및 단편화 패턴에 따라 자신 있게 할당됩니다.

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.

저자 스포트라이트: LC-TIMS-ToF MS/MS 및 PASEF를 통한 향상된 Histone PTM 이성질체 식별

액체 크로마토그래피, Trapped Ion Mobility Spectrometry 및 Time-Of-Flight Mass Spectrometry를 사용한 Histone Modification Screening

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.

상향식 단백질체학 워크플로우(그림 7)에는 일반적으로 미처리 샘플에서 표적 단백질을 추출한 다음 단백질의 농도를 정량화한 다음 일반적으로 겔 전기영동 또는 액체 크로마토그래피를 통한 분획이 포함됩니다. 분획 후, 단백질은 단백질 분해 효소(종종 트립신)를 사용하여 절단되고, 마지막으로 생성된 펩티드의 질량 분석 및 확립된 데이터베이스18을 ?...

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

액체 크로마토그래피, 포획 이온 이동도 분광법 및 비행 시간 질량 분석법(LC-TIMS-ToF MS/MS)을 기반으로 하는 분석 워크플로우는 주요 매개변수(머무름 시간[RT], 충돌 단면[CCS] 및 정확한 질량 대 전하[m/z] 비율)를 기반으로 히스톤 변형 및 식별에 대한 높은 신뢰도와 재현성 있는 "상향식" 분석을 제공합니다.

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

95 % 효율의 히스톤 추출 프로토콜을 사용하여 방법을 제안합니다. LC-TIMS-ToF MS/MS와 함께 짧은 시간에 히스톤에 존재하는 PTM에 대한 스크리닝을 얻을 수 있습니다. 그리고 무엇보다도, 이성질체를 분리할 수 있는 가능성.이성질체 펩타이드의 분리는 탠덤 질량분석법만으로는 가능하지만 종종 어렵습니다. TIMS를 통합함으로써 추가적인 차원의 분리를 도입하여 많은 위치 이성질체 식별을 단순화합니다. 또한 패시브 기술은 이러한 실험의 민감도를 높여 후성유전학적 변이를 분석하는 능력을 더욱 향상시킵니다.PTM 위치를 식별하는 능력이 향상됨에 따라 다양한 환경 또는 생물학적 요인 간의 상관 관계와 후성유전체에 미치는 영향을 추가로 결정할 수 있습니다. 히스톤 분석을 개선하고 촉진하는 방법은 의료 및 환경 화학을 포함한 여러 연구 분야에 적용될 수 있습니다. 네이티브 또는 손상된 상태에서의 단백질 확인의 역학은 HDX를 사용한 TIMS MS/MS의 구현을 통해 확립됩니다.LC-TIMS-ToF MS/MS 및 Nano LC-TIMS-ToF MS/MS를 통해 다양한 생체 시료에 존재하는 PTM의 차이를 차트로 작성합니다.

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).