Name: 作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定
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

本材料基于美国国家科学基金会（National Science Foundation）资助的工作。HRD-1547798 和授权号HRD-2111661。这些 NSF 赠款被授予佛罗里达国际大学，作为科学技术卓越研究中心 （CREST） 计划的一部分。这是佛罗里达国际大学环境研究所的第 1672 号贡献，这是佛罗里达国际大学的一项杰出项目。美国国立卫生研究院（National Institute of Health）根据拨款号提供了额外的支持。R21AI135469弗朗西斯科·费尔南德斯-利马（Francisco Fernandez-Lima）和格兰特·诺（Grant No.）R01HD106051本杰明·加西亚（Benjamin A. Garcia）以及美国国家科学基金会（National Science Foundation）的资助号。CHE-2127882 给 Benjamin A. Garcia。作者要感谢Mario Gomez Hernandez博士在初始方法开发过程中的初步支持。

注：组蛋白样品是使用改编自Bhanu等人（2020）12的方法提取的。
1. 样品制备
<ol><li>收获培养细胞<ol><li>当细胞汇合度达到 80% 时，使用台盼蓝排除确保它们存活。 注意：这些实验使用了HeLa S3细胞系，但这种方法可以应用于任何培养的细胞。</li>
		<li>吸出培养基，然后在每个板上涂抹 5 mL 1x 磷酸盐缓冲盐水 （PBS）。</li>
		<li>旋转板以冲洗所有?...

组蛋白组学分析和翻译后修饰表征

<ol>
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组蛋白是碱性蛋白质，通过以八聚体的形式与 DNA 相互作用来调节染色质结构，八聚体由四个核心组蛋白（H2A、H2B、H3 和 H4 各两个）组成）20。组蛋白含有许多赖氨酸和精氨酸残基，这些残基很容易被修饰，导致广泛的 PTM，通过影响组蛋白功能或与其他细胞蛋白结合来改变染色质化学21。PTM 可以通过协同工作来引发生物反应，在几种疾病中已经报道了特定的 PTM...

在真核细胞中，DNA 被包装成染色质，形成称为核小体的功能单元。这些单元由四个核心组蛋白（H2A、H2B、H3 和 H4 各两个）的八聚体组成1,2,3,4。组蛋白是真核生物中最丰富和高度保守的蛋白质之一，主要负责基因调控5.组蛋白翻译后修饰 （PTM） 在染色质动力学的调节和操纵各种生物过程（如 DNA 转录、复制和修复）中起着重要作用6。PTM 主要发生在与 DNA 3,7 接触的组蛋白 N 末端区域的可触及表面上。然而，尾部和核心修饰会影响染色质结构，改变核小体间相互作用并募集特定蛋白 3,8。
在基于液相色谱-质谱（LC-MS）的蛋白质组学中，当前的一个挑战是目标分析物的潜在共洗脱。在数据依赖性分析（DDA）的情况下，这意味着在MS/MS采集过程中，几个母离子的潜在损失9。飞行时间 （ToF） 仪器以非常高的频率 9,10（高达数十 kHz）11 获取频谱;这使得它们能够快速扫描复杂样品 （MS1） 中的总母离子，从而有望实现最佳灵敏度和 MS/MS 测序速率（高达 100 Hz）9，并使其成为生物样品分析的理想选择 10。然而，在这些高扫描速率下可用的灵敏度受到 MS/MS速率 9 的限制。结合捕获离子淌度谱（TIMS）和正交四极杆飞行时间（qToF）质谱仪，可以减轻这些局限性。在 TIMS 中，所有前驱离子都串联积累并洗脱作为其迁移率的函数，而不是选择具有四极杆9 的单个前驱体质量数。并行累积-串行碎片 （PASEF） 允许每秒处理数百个 MS/MS 事件，而不会损失任何灵敏度9。
这项工作的主要目的是展示 DDA 的最新发展，使用迁移阱中的平行积累，然后是顺序碎裂和碰撞诱导解离 （CID）。组蛋白 PTM 根据其保留时间 （RT）、迁移率和碎裂模式进行可靠分配。

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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>
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		<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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			<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>
			<td>28324</td>
			<td>NP-40 Surfact-Amps Detergent Solution</td>
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		<tr>
			<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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			<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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		<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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			<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>
			<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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			<td>Cell dissociation buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>13151014</td>
			<td></td>
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			<td>Ceramic scoring wafer</td>
			<td>Restek</td>
			<td>20116</td>
			<td></td>
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		<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

组蛋白在真核生物中高度丰富且保守，并且由于称为翻译后修饰 （PTM） 的结构，在基因调控中发挥着重要作用。参考外部或遗传因素确定每个 PTM 或 PTM 模式的位置和性质，可以使这些信息与生物反应（如 DNA 转录、复制或修复）进行统计相关。在本工作中，描述了一种用于检测生物样品中组蛋白PTMs的高通量分析方案。使用互补液相色谱法、俘获离子淌度谱法和飞行时间质谱法 （LC-TIMS-ToF MS/MS） 可以在单次分析中分离和分配最具生物学相关性的修饰。所描述的方法利用了相关数据采集 （DDA） 的最新发展，使用迁移阱中的平行积累，然后是顺序碎片化和碰撞诱导的解离。组蛋白 PTM 根据其保留时间、迁移率和碎片模式进行可靠分配。

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.

作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

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.

自下而上的蛋白质组学工作流程（图7）通常包括以下内容：从粗样品中提取目标蛋白质，然后定量蛋白质的浓度，然后进行分离，通常通过凝胶电泳或液相色谱法进行分馏。分馏后，使用蛋白水解酶（通常是胰蛋白酶）消化蛋白质，最后，使用已建立的数据库18对所得肽进行质谱分析和蛋白质鉴定。序列信息来自所示质荷比 （m/z） 范围内的前驱离?...

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

基于液相色谱、俘获离子淌度谱和飞行时间质谱 （LC-TIMS-ToF MS/MS） 的分析工作流程，用于基于主要参数（保留时间 [RT]、碰撞截面 [CCS] 和准确的质荷比 [m/z] 比）对组蛋白修饰和鉴定进行高可信度和高度可重复性的“自下而上”分析。

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

提出了一种使用组蛋白提取方案的方法，效率为95%。与LC-TIMS-ToF MS/MS一起，可以在短时间内筛选组蛋白中存在的PTM。最重要的是，分离异构体的可能性。异构肽的分离虽然可能单独使用串联质谱法，但通常很困难。通过结合TIMS，我们引入了一个额外的分离维度，简化了许多位置异构体的鉴定。此外，被动技术提高了这些实验的灵敏度，进一步提高了我们分析表观遗传变异的能力。随着识别PTM位置的能力增强，能够进一步确定各种环境或生物因素之间的相关性，以及它们对表观基因组的影响。改进和促进组蛋白分析的方法可应用于多个研究领域，包括医学和环境化学。通过使用 HDX 实施 TIMS MS/MS 将建立天然或受损状态下的蛋白质确认动力学。通过LC-TIMS-ToF MS/MS和Nano LC-TIMS-ToF MS/MS，绘制了各种生物样品中PTM的差异。

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).

作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定

作者聚焦：通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定