Name: 著者スポットライト:LC-TIMS-ToF、MS/MS、およびPASEFによるヒストンPTM異性体の同定の強化(英語)
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

この資料は、助成金番号の下で全米科学財団が支援する研究に基づいています。HRD-1547798およびグラント番号HRD-2111661 です。これらのNSF助成金は、科学技術研究センター(CREST)プログラムの一環としてフロリダ国際大学に授与されました。これは、フロリダ国際大学の卓越したプログラムである環境研究所からの寄稿番号1672です。米国国立衛生研究所(NIH)は、助成金No.フランシスコ・フェルナンデス・リマとグラントNo.にR21AI135469。ベンジャミン・A・ガルシア(Benjamin A. Garcia)にR01HD106051され、全米科学財団(National Science Foundation)から助成金番号で授与された。CHE-2127882 から Benjamin A. Garcia 宛て。著者らは、分析法開発の初期における Mario Gomez Hernandez 博士の初期サポートに感謝します。

注:ヒストンサンプルは、Bhanu et al.(2020)12から採用された方法を使用して抽出されました。
1. サンプル調製
<ol><li>培養細胞の回収<ol><li>細胞が80%コンフルエントになったら、トリパンブルー排除法を用いて細胞が生存可能であることを確認します。 注:これらの実験にはHeLa S3細胞株を使用しましたが、この方法はあらゆる培養細胞に適用?...

ヒストンプロテオミクス解析と翻訳後修飾特性評価

<ol>
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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. 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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. 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ヒストンは、4つのコアヒストン(H2A、H2B、H3、H4の各2つ)からなる八量体の形でDNAと相互作用することにより、クロマチン構造を調節する基本的なタンパク質です20。ヒストンには多数のリジン残基とアルギニン残基が含まれており、これらは容易に修飾され、ヒストン機能に影響を与えたり、他の細胞タンパク質に結合したりしてクロマチンの化学的性質を変化させる広範な...

真核細胞では、DNAはクロマチンとしてヌクレオソームと呼ばれる機能単位にパッケージ化されています。これらのユニットは、4つのコアヒストン(H2A、H2B、H3、H4の各2つ)の八量体で構成されています1,2,3,4。ヒストンは、真核生物の中で最も豊富で保存性の高いタンパク質の一つであり、遺伝子調節に大きく関与しています5。ヒストンの翻訳後修飾(PTM)は、クロマチン動態の調節に大きな役割を果たし、DNAの転写、複製、修復などのさまざまな生物学的プロセスに関与しています6。PTMは、主にDNAと接触しているヒストンのN末端領域のアクセス可能な表面に発生します3,7。しかし、尾部とコアの修飾はクロマチン構造に影響を与え、ヌクレオソーム間の相互作用を変化させ、特定のタンパク質を動員します3,8。
液体クロマトグラフィー質量分析(LC-MS)ベースのプロテオミクスにおける現在の課題は、目的の分析種の共溶出の可能性です。データ依存性分析(DDA)の場合、これはMS/MS取り込みプロセス中にいくつかのプリカーサーイオンが失われる可能性があることを意味します9。飛行時間型(ToF)機器は、非常に高い周波数9,10(最大数十kHz)でスペクトルを取得します11。これにより、複合体サンプル(MS1)内の全プリカーサーイオンを迅速にスキャンできるため、最適な感度とMS/MSシーケンシングレート(最大100 Hz)が約束され9、生物学的サンプル分析に理想的です10。それにもかかわらず、これらの高いスキャンレートで利用可能な感度は、MS/MSレート9によって制限されます。これらの制限を軽減するために、直交四重極飛行時間型(qToF)質量分析計と組み合わせたトラップドイオンモビリティースペクトロメトリー(TIMS)の追加が使用されました。TIMSでは、すべての前駆体イオンは、四重極9で単一の前駆体質量を選択するのではなく、それらの移動度の関数としてタンデムで蓄積され、溶出されます。PASEF(Parallel Accumulation-Serial Fragmentation)により、感度を損なうことなく、毎秒数百のMS/MSイベントを処理できます9。
この研究の主な目的は、移動トラップでの並列蓄積とそれに続く連続的な断片化と衝突誘起解離(CID)を使用して、DDAの最近の発展を示すことでした。Histone PTM は、保持時間(RT)、移動度、およびフラグメンテーションパターンに基づいて、自信を持って割り当てました。

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			<td>-80 &#176;C Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td>Animal Origin-Free</td>
		</tr>
		<tr>
			<td>1 mL Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060710</td>
			<td>Finntip Flex 1000 &#956;L, nonsterile, nonfiltered, racked tips</td>
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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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		<tr>
			<td>10 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060100</td>
			<td>Finntip Flex, 10 &#956;L, nonsterile, non-filtered, racked</td>
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			<td>10% NP-40</td>
			<td>Thermo Fisher Scientific</td>
			<td>28324</td>
			<td>NP-40 Surfact-Amps Detergent Solution</td>
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			<td>10x Dulbecco&#8217;s PBS without Ca2+/Mg2+</td>
			<td>(Mediatech)</td>
			<td>MT21031CM</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Corning</td>
			<td>352196</td>
			<td>Falcon Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>200 &#181;L Gel-Loading Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-707-138</td>
			<td>Fisherbrand&#160;Gel-Loading Tips, 1&#8211;200 &#956;L</td>
		</tr>
		<tr>
			<td>200 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
			<td>94060310</td>
			<td>Finntip Flex 200&#956;L, nonsterile, nonfiltered, racked tips</td>
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		<tr>
			<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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		<tr>
			<td>50 mL Conical Tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td>Falcon Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>96-well flat bottom plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565501</td>
			<td></td>
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		<tr>
			<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>
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			<td>Acetonitrile (ACN)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A998</td>
			<td>HPLC, Fisher Chemical</td>
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		<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>
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		<tr>
			<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>
			<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>
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		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Heat shock fraction, pH 7, &#8805;98%</td>
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			<td>Calcium chloride, CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>Anhydrous, powder, &#8805;97%</td>
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		<tr>
			<td>Cell dissociation buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>13151014</td>
			<td></td>
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		<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

ヒストンタンパク質は真核生物に豊富に存在し、保存されており、翻訳後修飾(PTM)として知られる構造の結果として、遺伝子制御に大きな役割を果たしています。外的要因または遺伝的要因を参照して、各PTMの位置と性質またはPTMのパターンを特定することで、この情報をDNA転写、複製、修復などの生物学的応答と統計的に相関させることができます。本研究では、生体試料からヒストンPTMを検出するためのハイスループット分析プロトコルについて説明します。相補的な液体クロマトグラフィー、トラップドイオンモビリティースペクトロメトリー、飛行時間型質量分析(LC-TIMS-ToF MS/MS)を使用することで、1 回の分析で最も生物学的に関連性の高い修飾を分離し、PTM に割り当てることができます。説明されているアプローチは、モビリティトラップでの並列蓄積とそれに続く連続的なフラグメンテーションと衝突による解離を使用した依存データ収集(DDA)の最近の開発を利用しています。Histone PTMは、保持時間、移動度、フラグメンテーションパターンに基づいて確実に割り当てられます。

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.

著者スポットライト:LC-TIMS-ToF、MS/MS、およびPASEFによるヒストンPTM異性体の同定の強化(英語)

液体クロマトグラフィー、トラップ型イオンモビリティー分光分析、飛行時間型質量分析を用いたヒストン修飾スクリーニング(英語)

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.

ボトムアッププロテオミクスワークフロー(図7)では、通常、粗サンプルから標的タンパク質を抽出し、タンパク質の濃度を定量し、通常はゲル電気泳動または液体クロマトグラフィーで分画します。分画後、タンパク質はタンパク質分解酵素(しばしばトリプシン)を用いて消化され、最後に、得られたペプチドの質量分析および確立されたデータベースを用いたタンパ?...

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

液体クロマトグラフィー、トラップ型イオンモビリティー分光、飛行時間型質量分析(LC-TIMS-ToF MS/MS)に基づく分析ワークフローにより、主要パラメーター(保持時間[RT]、衝突断面積[CCS]、正確な質量電荷比[m/z])に基づくヒストン修飾および同定の高信頼性で再現性の高い「ボトムアップ」分析を実現します。

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%の効率でヒストン抽出プロトコルを使用する方法を提案します。LC-TIMS-ToF MS/MS と併用することで、ヒストン中に存在する PTM のスクリーニングを短時間で得ることができます。そして何よりも、異性体を分離する可能性。異性体ペプチドの分離は、タンデム質量分析のみを使用しても可能ですが、多くの場合、困難です。TIMSを組み込むことで、分離の次元がさらに向上し、多くの位置異性体同定が簡素化されます。さらに、パッシブテクノロジーはこれらの実験の感度を高め、エピジェネティックな変異を分析する能力をさらに向上させます。PTMの位置を特定する能力が高まると、さまざまな環境的または生物学的要因と、それらがエピゲノムに及ぼす影響との相関関係をさらに決定できるようになります。ヒストンの分析を改善および促進する方法は、医学化学や環境化学など、いくつかの研究分野に適用できます。天然状態または損傷状態におけるタンパク質確認のダイナミクスは、HDXによるTIMS MS/MSの実装から確立されます。LC-TIMS-ToF MS/MSおよびNano LC-TIMS-ToF MS/MSにより、さまざまな生体試料に存在するPTMの違いをグラフ化します。

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).