Name: Autor im Rampenlicht: Verbesserte Identifizierung von Histon-PTM-Isomeren durch LC-TIMS-TOF MS/MS und 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

Dieses Material basiert auf Arbeiten, die von der National Science Foundation unter der Fördernummer unterstützt werden. HRD-1547798 und Grant No. HRD-2111661. Diese NSF-Zuschüsse wurden der Florida International University im Rahmen des CREST-Programms (Centers of Research Excellence in Science and Technology) gewährt. Dies ist der Beitrag Nummer 1672 des Institute of Environment, einem herausragenden Programm an der Florida International University. Zusätzliche Unterstützung wurde vom National Institute of Health im Rahmen des Grant No. R21AI135469 an Francisco Fernandez-Lima und Grant No. R01HD106051 an Benjamin A. Garcia sowie von der National Science Foundation unter Grant No. CHE-2127882 an Benjamin A. Garcia. Die Autoren danken Dr. Mario Gomez Hernandez für die anfängliche Unterstützung bei der ersten Methodenentwicklung.

HINWEIS: Histonproben wurden mit einer Methode extrahiert, die von Bhanu et al. (2020)12 übernommen wurde.
1. Probenvorbereitung
<ol><li>Ernte von kultivierten Zellen<ol><li>Wenn Zellen zu 80 % konfluent sind, stellen Sie sicher, dass sie lebensfähig sind, indem Sie den Trypanblau-Ausschluss verwenden. HINWEIS: Für diese Experimente wurde eine HeLa S3-Zelllinie verwendet, aber diese Methode kann auf alle kultivierten Zellen angewendet werden.</l...

Histon-Proteomanalyse und posttranslationale Modifikationscharakterisierung

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Histone sind grundlegende Proteine, die die Chromatinstruktur regulieren, indem sie mit der DNA in Form von Oktameren interagieren, die aus den vier Kernhistonen (je zwei von H2A, H2B, H3 und H4) bestehen20. Histone enthalten zahlreiche Lysin- und Argininreste, die leicht modifiziert werden können, was zu umfangreichen PTMs führt, die die Chromatinchemie verändern, indem sie die Histonfunktion beeinflussen oder an andere zelluläre Proteine binden21. PTMs können biologi...

In eukaryotischen Zellen wird DNA als Chromatin in funktionelle Einheiten verpackt, die Nukleosomen genannt werden. Diese Einheiten bestehen aus einem Oktamer von vier Kernhistonen (je zwei von H2A, H2B, H3 und H4)1,2,3,4. Histone gehören zu den am häufigsten vorkommenden und am höchsten konservierten Proteinen in Eukaryoten, die maßgeblich für die Genregulation verantwortlich sind5. Posttranslationale Histonmodifikationen (PTMs) spielen eine große Rolle bei der Regulation der Chromatindynamik und steuern verschiedene biologische Prozesse wie DNA-Transkription, -Replikation und -Reparatur6. PTMs treten hauptsächlich auf der zugänglichen Oberfläche der N-terminalen Regionen von Histonen auf, die mit DNA in Kontakt stehen 3,7. Schwanz- und Kernmodifikationen beeinflussen jedoch die Chromatinstruktur, verändern die Internukleosomeninteraktionen und rekrutieren spezifische Proteine 3,8.
Eine aktuelle Herausforderung bei der Flüssigchromatographie-Massenspektrometrie (LC-MS)-basierten Proteomik ist die potenzielle Co-Elution von Analyten von Interesse. Bei datenabhängigen Analysen (DDA) bedeutet dies den potenziellen Verlust mehrerer Vorläuferionen während des MS/MS-Erfassungsprozesses9. Time-of-Flight (ToF)-Instrumente erfassen Spektren mit sehr hoher Frequenz 9,10 (bis zu zehn kHz)11; Dadurch sind sie in der Lage, die gesamten Vorläuferionen innerhalb einer komplexen Probe (MS1) schnell zu scannen, was eine optimale Empfindlichkeit und MS/MS-Sequenzierungsraten (bis zu 100 Hz)9 verspricht und sie ideal für die biologische Probenanalysemacht 10. Die Empfindlichkeit, die bei diesen hohen Scanraten verfügbar ist, ist jedoch durch die MS/MS-Rate9 begrenzt. Die Hinzufügung der Gefangenen-Ionen-Mobilitätsspektrometrie (TIMS) in Kombination mit einem orthogonalen Quadrupol-Flugzeitspektrometer (qToF) wurde verwendet, um diese Einschränkungen zu mildern. In TIMS werden alle Vorläuferionen im Tandem akkumuliert und in Abhängigkeit von ihrer Mobilität eluiert, anstatt einzelne Vorläufermassen mit einem Quadrupol9 auszuwählen. Die parallele Akkumulations-serielle Fragmentierung (PASEF) ermöglicht Hunderte von MS/MS-Ereignissen pro Sekunde ohne Verlust der Empfindlichkeit9.
Das Hauptziel dieser Arbeit war es, die jüngsten Entwicklungen der DDA unter Verwendung von paralleler Akkumulation in der Mobilitätsfalle, gefolgt von sequentieller Fragmentierung und kollisionsinduzierter Dissoziation (CID), aufzuzeigen. Histon-PTMs wurden basierend auf ihren Retentionszeiten (RTs), Mobilitäten und Fragmentierungsmustern sicher zugewiesen.

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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>
		</tr>
		<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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		<tr>
			<td>10% NP-40</td>
			<td>Thermo Fisher Scientific</td>
			<td>28324</td>
			<td>NP-40 Surfact-Amps Detergent Solution</td>
		</tr>
		<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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		<tr>
			<td>2x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610737</td>
			<td>Premixed protein sample buffer for SDS-PAGE</td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>96-well plate, V-Bottom 600 &#956;L</td>
			<td>Axygen</td>
			<td>P-DW-500-C-S</td>
			<td></td>
		</tr>
		<tr>
			<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>
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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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		<tr>
			<td>Ammonium bicarbonate, NH4HCO3</td>
			<td>Sigma Aldrich</td>
			<td>09830</td>
			<td>BioUltra, &#8805;99.5% (T)</td>
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		<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>
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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>
		</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

Histonproteine sind bei Eukaryoten sehr häufig und konserviert und spielen aufgrund von Strukturen, die als posttranslationale Modifikationen (PTMs) bekannt sind, eine große Rolle bei der Genregulation. Die Identifizierung der Position und Art jedes PTMs oder Musters von PTMs in Bezug auf externe oder genetische Faktoren ermöglicht es, diese Informationen statistisch mit biologischen Reaktionen wie DNA-Transkription, Replikation oder Reparatur zu korrelieren. In der vorliegenden Arbeit wird ein Hochdurchsatz-Analyseprotokoll für den Nachweis von Histon-PTMs aus biologischen Proben beschrieben. Der Einsatz von komplementärer Flüssigkeitschromatographie, Ionenmobilitätsspektrometrie und Flugzeit-Massenspektrometrie (LC-TIMS-ToF MS/MS) ermöglicht die Trennung und PTM-Zuordnung der biologisch relevantesten Modifikationen in einer einzigen Analyse. Der beschriebene Ansatz nutzt die jüngsten Entwicklungen in der abhängigen Datenerfassung (DDA) mit paralleler Akkumulation in der Mobilitätsfalle, gefolgt von sequentieller Fragmentierung und kollisionsinduzierter Dissoziation. Histon-PTMs werden basierend auf ihrer Retentionszeit, Mobilität und ihrem Fragmentierungsmuster sicher zugewiesen.

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 im Rampenlicht: Verbesserte Identifizierung von Histon-PTM-Isomeren durch LC-TIMS-TOF MS/MS und PASEF

Histonmodifikations-Screening mittels Flüssigkeitschromatographie, Ionenmobilitätsspektrometrie und Flugzeit-Massenspektrometrie

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.

Ein Bottom-up-Proteom-Workflow (Abbildung 7) umfasst in der Regel Folgendes: Extraktion des Zielproteins aus einer Rohprobe, gefolgt von der Quantifizierung der Konzentration des Proteins/der Proteine und der anschließenden Fraktionierung, in der Regel durch Gelelektrophorese oder Flüssigkeitschromatographie. Nach der Fraktionierung werden die Proteine mit einem proteolytischen Enzym (oft Trypsin) verdaut und schließlich eine massenspektrometrische Analyse der resultierenden Peptide und e...

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

Ein analytischer Arbeitsablauf, der auf Flüssigkeitschromatographie, Mobilitätsspektrometrie mit gefangenen Ionen und Flugzeit-Massenspektrometrie (LC-TIMS-ToF MS/MS) basiert, für eine hochzuverlässige und hochgradig reproduzierbare "Bottom-up"-Analyse von Histonmodifikationen und -identifizierung basierend auf Hauptparametern (Retentionszeit [RT], Kollisionsquerschnitt [CCS] und genaues Masse-zu-Ladungs-Verhältnis [m/z]).

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

Es wird eine Methode vorgeschlagen, bei der ein Histon-Extraktionsprotokoll mit einer Effizienz von 95 % verwendet wird. Zusammen mit LC-TIMS-ToF MS/MS ist es möglich, in kurzer Zeit ein Screening auf die im Histon vorhandenen PTMs zu erhalten. Und vor allem die Möglichkeit, Isomere zu trennen.Die Trennung von isomeren Peptiden ist zwar nur mit Tandem-Massenspektrometrie möglich, aber oft schwierig. Durch die Integration von TIMS führen wir eine zusätzliche Dimension der Trennung ein und vereinfachen viele Positionsisomerenidentifikationen. Darüber hinaus erhöht die passive Technologie die Empfindlichkeit dieser Experimente und verbessert so unsere Fähigkeit, epigenetische Variationen zu analysieren.Mit der verbesserten Fähigkeit, PTM-Positionen zu identifizieren, geht die Möglichkeit einher, die Korrelation zwischen verschiedenen Umwelt- oder biologischen Faktoren und deren Auswirkungen auf das Epigenom weiter zu bestimmen. Methoden, die die Analyse von Histonen verbessern und erleichtern, können auf verschiedene Forschungsbereiche angewendet werden, einschließlich der Medizin- und Umweltchemie. Die Dynamik der Proteinbestätigung in nativen oder beschädigten Zuständen wird durch die Implementierung von TIMS MS/MS mit HDX ermittelt.Durch LC-TIMS-ToF MS/MS und Nano LC-TIMS-ToF MS/MS werden die Unterschiede in PTM in verschiedenen biologischen Proben kartiert.

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).