Name: Yazar Spotlight: LC-TIMS-ToF MS/MS ve PASEF aracılığıyla gelişmiş histon PTM izomer tanımlaması
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

Bu materyal, Ulusal Bilim Vakfı tarafından Hibe No kapsamında desteklenen çalışmalara dayanmaktadır. HRD-1547798 ve Hibe No. HRD-2111661. Bu NSF Hibeleri, Bilim ve Teknolojide Araştırma Mükemmeliyet Merkezleri (CREST) Programının bir parçası olarak Florida Uluslararası Üniversitesi'ne verildi. Bu, Florida Uluslararası Üniversitesi'nde Seçkin Bir Program olan Çevre Enstitüsü'nden 1672 numaralı katkıdır. Ulusal Sağlık Enstitüsü tarafından Hibe No. kapsamında ek destek sağlanmıştır. R21AI135469 Francisco Fernandez-Lima ve Grant No. R01HD106051 Benjamin A. Garcia'ya ve Ulusal Bilim Vakfı tarafından Hibe No. CHE-2127882'den Benjamin A. Garcia'ya. Yazarlar, ilk yöntem geliştirmeleri sırasında Dr. Mario Gomez Hernandez'in ilk desteğini kabul etmek isterler.

NOT: Histon örnekleri, Bhanu ve ark. (2020)12.
1. Numune hazırlama
<ol><li>Kültürlenmiş hücrelerin toplanması<ol><li>Hücreler %80 birleştiğinde, tripan mavisi dışlamasını kullanarak canlı olduklarından emin olun. NOT: Bu deneyler için bir HeLa S3 hücre hattı kullanıldı, ancak bu yöntem herhangi bir kültürlenmiş hücreye uygulanabilir.</li>
		<li>Ortamı aspire edin, ardından her plakaya 5 mL 1x fosfat tamponlu salin (PBS) ...

Histon Proteomik Analizi ve Translasyon Sonrası Modifikasyon Karakterizasyonu

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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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Histonlar, dört çekirdek histondan (H2A, H2B, H3 ve H4'ün ikişer tane) oluşan oktamerler şeklinde DNA ile etkileşime girerek kromatin yapısını düzenleyen temel proteinlerdir.20. Histonlar, kolayca değiştirilebilen çok sayıda lizin ve arginin kalıntısı içerir, bu da histon fonksiyonunu etkileyerek veya diğer hücresel proteinlere bağlanarak kromatin kimyasını değiştiren kapsamlı PTM'lere yol açar21. PTM'ler, çeşitli hastalıklarda, özellikle de ...

Ökaryotik hücrelerde DNA, kromatin olarak nükleozom adı verilen fonksiyonel birimler halinde paketlenir. Bu birimler dört çekirdek histondan oluşan bir oktamerden oluşur (her biri H2A, H2B, H3 ve H4'ten ikişer)1,2,3,4. Histonlar, gen regülasyonundan büyük ölçüde sorumlu olan ökaryotlarda en bol bulunan ve yüksek oranda korunan proteinler arasındadır5. Histon posttranslasyonel modifikasyonları (PTM'ler), kromatin dinamiğinin düzenlenmesinde büyük rol oynar ve DNA transkripsiyonu, replikasyonu ve onarımı gibi çeşitli biyolojik süreçlerin donatılmasında büyük rol oynar6. PTM'ler öncelikle DNA 3,7 ile temas halinde olan histonların N-terminal bölgelerinin erişilebilir yüzeyinde meydana gelir. Bununla birlikte, kuyruk ve çekirdek modifikasyonları kromatin yapısını etkiler, nükleozomlar arası etkileşimleri değiştirir ve spesifik proteinleri işe alır 3,8.
Sıvı kromatografi-kütle spektrometrisi (LC-MS) tabanlı proteomik sırasındaki güncel bir zorluk, ilgilenilen analitlerin potansiyel birlikte elüsyonudur. Veriye bağlı analizler (DDA) söz konusu olduğunda, bu, MS/MS edinme işlemi sırasında birkaç öncü iyonun potansiyel kaybına dönüşür9. Uçuş süresi (ToF) cihazları çok yüksek frekanstaspektrum alır 9,10 (onlarca kHz'e kadar)11; bu, karmaşık bir numune (MS1) içindeki toplam öncü iyonları hızlı bir şekilde tarayabilmelerini sağlar, böylece optimum hassasiyet ve MS/MS dizileme oranları (100 Hz'e kadar)9 vaat eder ve onları biyolojik numune analizi için ideal hale getirir10. Bununla birlikte, bu yüksek tarama hızlarında mevcut olan hassasiyet, MS/MS hızı9 ile sınırlıdır. Bu sınırlamaları azaltmak için ortogonal dört kutuplu uçuş süresi (qToF) kütle spektrometresi ile birlikte tuzağa düşürülmüş iyon hareketlilik spektrometrisinin (TIMS) eklenmesi kullanıldı. TIMS'de, tüm öncü iyonlar art arda biriktirilir ve dört kutuplu9 ile tek öncü kütleleri seçmek yerine, hareketliliklerinin bir fonksiyonu olarak ayrıştırılır. Paralel birikim-seri parçalanma (PASEF), herhangi bir hassasiyet kaybı olmadan saniyede yüzlerce MS/MS olayına izin verir9.
Bu çalışmanın temel amacı, hareketlilik tuzağında paralel birikim ve ardından sıralı parçalanma ve çarpışma kaynaklı ayrışma (CID) kullanarak DDA'nın son gelişmelerini göstermekti. Histon PTM'leri, tutma sürelerine (RT'ler), hareketliliklerine ve parçalanma modellerine göre güvenle atandı.

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		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<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>
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		<tr>
			<td>Acetone</td>
			<td>Sigma Aldrich</td>
			<td>179124</td>
			<td>ACS reagent, &#8805;99.5%</td>
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		<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>
		</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>
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		<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

Histon proteinleri ökaryotlar arasında oldukça bol miktarda bulunur ve korunur ve posttranslasyonel modifikasyonlar (PTM'ler) olarak bilinen yapıların bir sonucu olarak gen düzenlemesinde büyük rol oynar. Dış veya genetik faktörlere göre her bir PTM'nin veya PTM modelinin konumunu ve doğasını belirlemek, bu bilginin DNA transkripsiyonu, replikasyonu veya onarımı gibi biyolojik tepkilerle istatistiksel olarak ilişkilendirilmesine izin verir. Bu çalışmada, biyolojik örneklerden histon PTM'lerinin tespiti için yüksek verimli bir analitik protokol açıklanmaktadır. Tamamlayıcı sıvı kromatografisi, sıkışmış iyon hareketlilik spektrometrisi ve uçuş zamanı kütle spektrometrisinin (LC-TIMS-ToF MS/MS) kullanımı, biyolojik olarak en ilgili modifikasyonların tek bir analizde ayrılmasını ve PTM atanmasını sağlar. Açıklanan yaklaşım, hareketlilik tuzağında paralel birikim ve ardından sıralı parçalanma ve çarpışma kaynaklı ayrışma kullanarak bağımlı veri toplamadaki (DDA) son gelişmelerden yararlanır. Histon PTM'leri, tutma sürelerine, hareketliliklerine ve parçalanma modellerine göre güvenle atanır.

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.

Yazar Spotlight: LC-TIMS-ToF MS/MS ve PASEF aracılığıyla gelişmiş histon PTM izomer tanımlaması

Sıvı Kromatografisi, Sıkışmış İyon Hareketlilik Spektrometrisi ve Uçuş Süresi Kütle Spektrometrisi Kullanılarak Histon Modifikasyon Taraması

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.

Aşağıdan yukarıya bir proteomik iş akışı (Şekil 7) tipik olarak aşağıdakileri içerir: ham bir numuneden hedef protein(ler)in ekstraksiyonu, ardından protein(ler)in konsantrasyonunun ölçülmesi ve ardından genellikle jel elektroforezi veya sıvı kromatografisi ile fraksiyonlama. Fraksiyonlamadan sonra, proteinler bir proteolitik enzim (genellikle tripsin) kullanılarak sindirilir ve son olarak, elde edilen peptitlerin kütle spektrometrik analizi ve yerleşik bir veri taban?...

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

Histon modifikasyonlarının yüksek güvenilirlikli ve yüksek oranda tekrarlanabilir "aşağıdan yukarıya" analizi ve temel parametrelere (alıkonma süresi [RT], çarpışma kesiti [CCS] ve doğru kütle-yük [m/z] oranı) dayalı tanımlama için sıvı kromatografisi, sıkışmış iyon hareketlilik spektrometrisi ve uçuş zamanı kütle spektrometrisine (LC-TIMS-ToF MS/MS) dayalı analitik bir iş akışı.

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 verimlilikle histon ekstraksiyon protokolü kullanılarak bir yöntem önerilmiştir. LC-TIMS-ToF MS/MS ile birlikte, histonda bulunan PTM'ler için kısa sürede bir tarama elde etmek mümkündür. Ve hepsinden önemlisi, izomerleri ayırma imkanı.İzomerik peptitlerin ayrılması, tek başına tandem kütle spektrometrisi kullanılarak mümkün olsa da, genellikle zordur. TIMS'i dahil ederek, birçok konumsal izomer tanımlamasını basitleştirerek ekstra bir ayırma boyutu sunuyoruz. Ek olarak, pasif teknoloji bu deneylerin hassasiyetini artırarak epigenetik varyasyonları analiz etme yeteneğimizi daha da geliştirir.PTM pozisyonlarını belirleme yeteneğinin artmasıyla, çeşitli çevresel veya biyolojik faktörler arasındaki korelasyonu ve bunların epigenom üzerindeki etkilerini daha fazla belirleme yeteneği ortaya çıkar. Histonların analizini iyileştiren ve kolaylaştıran yöntemler, tıbbi ve çevre kimyası dahil olmak üzere çeşitli araştırma alanlarına uygulanabilir. Doğal veya hasarlı durumlarda protein doğrulamasının dinamikleri, TIMS MS/MS'nin HDX ile uygulanmasından belirlenecektir.LC-TIMS-ToF MS/MS ve Nano LC-TIMS-ToF MS/MS ile çeşitli biyolojik örneklerde bulunan PTM'deki farklılıklar grafiklendirilecektir.

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).