Name: Autore in primo piano: Identificazione migliorata degli isomeri PTM istonici tramite LC-TIMS-ToF MS/MS e 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

Questo materiale si basa sul lavoro sostenuto dalla National Science Foundation nell'ambito della sovvenzione n. HRD-1547798 e Grant No. HRD-2111661. Queste sovvenzioni NSF sono state assegnate alla Florida International University nell'ambito del programma CREST (Centers of Research Excellence in Science and Technology). Questo è il contributo numero 1672 dell'Institute of Environment, un programma preminente presso la Florida International University. Un ulteriore sostegno è stato fornito dal National Institute of Health nell'ambito della sovvenzione n. R21AI135469 a Francisco Fernandez-Lima e Grant No. R01HD106051 a Benjamin A. Garcia, così come dalla National Science Foundation nell'ambito della sovvenzione n. CHE-2127882 a Benjamin A. Garcia. Gli autori desiderano riconoscere il supporto iniziale del Dr. Mario Gomez Hernandez durante gli sviluppi iniziali del metodo.

NOTA: I campioni di istoni sono stati estratti utilizzando un metodo adattato da Bhanu et al. (2020)12.
1. Preparazione del campione
<ol><li>Raccolta di cellule in coltura<ol><li>Quando le cellule sono confluenti all'80%, assicurarsi che siano vitali utilizzando l'esclusione del blu di tripano. NOTA: Per questi esperimenti è stata utilizzata una linea cellulare HeLa S3, ma questo metodo può essere applicato a qualsiasi cellula in coltura.</li>
		...

Analisi proteomica istonica e caratterizzazione delle modificazioni post-traduzionali

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Gli istoni sono proteine di base che regolano la struttura della cromatina interagendo con il DNA sotto forma di ottameri costituiti dai quattro istoni principali (due ciascuno di H2A, H2B, H3 e H4)20. Gli istoni contengono numerosi residui di lisina e arginina, che vengono prontamente modificati, portando a PTM estesi che alterano la chimica della cromatina influenzando la funzione degli istoni o legandosi ad altre proteine cellulari21. I PTM possono suscitare risposte bio...

Nelle cellule eucariotiche, il DNA è impacchettato come cromatina in unità funzionali chiamate nucleosomi. Queste unità sono composte da un ottamero di quattro istoni centrali (due ciascuno di H2A, H2B, H3 e H4)1,2,3,4. Gli istoni sono tra le proteine più abbondanti e altamente conservate negli eucarioti, che sono in gran parte responsabili della regolazione genica5. Le modificazioni post-traduzionali degli istoni (PTM) svolgono un ruolo importante nella regolazione della dinamica della cromatina e nella gestione di vari processi biologici come la trascrizione, la replicazione e la riparazione del DNA6. I PTM si verificano principalmente sulla superficie accessibile delle regioni N-terminali degli istoni che sono in contatto con il DNA 3,7. Tuttavia, le modificazioni della coda e del core influenzano la struttura della cromatina, alterando le interazioni internucleosomiche e reclutando proteine specifiche 3,8.
Una sfida attuale durante la proteomica basata sulla cromatografia liquida-spettrometria di massa (LC-MS) è la potenziale co-eluizione degli analiti di interesse. Nel caso di analisi data-dependent (DDA), ciò si traduce nella potenziale perdita di diversi ioni precursori durante il processo di acquisizione MS/MS9. Gli strumenti a tempo di volo (ToF) acquisiscono spettri ad altissima frequenza 9,10 (fino a decine di kHz)11; questo li rende in grado di scansionare rapidamente gli ioni precursori totali all'interno di un campione complesso (MS1), promettendo così una sensibilità ottimale e velocità di sequenziamento MS/MS (fino a 100 Hz)9 e rendendoli ideali per l'analisi di campioni biologici10. Ciononostante, la sensibilità disponibile a queste elevate velocità di scansione è limitata dalla velocità MS/MS9. Per mitigare queste limitazioni è stata utilizzata l'aggiunta della spettrometria di mobilità ionica intrappolata (TIMS) in combinazione con uno spettrometro di massa a tempo di volo (qToF) a quadrupolo ortogonale. In TIMS, tutti gli ioni precursori sono accumulati in tandem ed eluiti in funzione della loro mobilità, piuttosto che selezionare singole masse precursori con un quadrupolo9. La frammentazione seriale ad accumulo parallela (PASEF) consente centinaia di eventi MS/MS al secondo senza alcuna perdita di sensibilità9.
L'obiettivo principale di questo lavoro è stato quello di mostrare i recenti sviluppi della DDA utilizzando l'accumulo parallelo nella trappola della mobilità seguito dalla frammentazione sequenziale e dalla dissociazione indotta da collisione (CID). I PTM istonici sono stati assegnati con sicurezza in base ai loro tempi di ritenzione (RT), mobilità e modelli di frammentazione.

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			<td>-80 &#176;C Freezer</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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			<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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			<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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		<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>
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			<td>200 &#181;L Pipette Tips</td>
			<td>Thermo Fisher Scientific</td>
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			<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>
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			<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>
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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>
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			<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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			<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>
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			<td>Ceramic scoring wafer</td>
			<td>Restek</td>
			<td>20116</td>
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			<td>Compass DataAnalysis 6.0</td>
			<td>Bruker Datonics</td>
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			<td>Compass HyStar 6.2</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Compass IsotopePattern</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Compass timsControl 4.1</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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			<td>Coomassie Brilliant Blue R-250</td>
			<td>Bio-Rad</td>
			<td>1610436</td>
			<td></td>
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			<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

Le proteine istoniche sono molto abbondanti e conservate tra gli eucarioti e svolgono un ruolo importante nella regolazione genica come risultato di strutture note come modificazioni post-traduzionali (PTM). Identificare la posizione e la natura di ogni PTM o pattern di PTM in riferimento a fattori esterni o genetici consente di correlare statisticamente queste informazioni con risposte biologiche come la trascrizione, la replicazione o la riparazione del DNA. Nel presente lavoro, viene descritto un protocollo analitico ad alto rendimento per la rilevazione di PTM istonici da campioni biologici. L'uso della cromatografia liquida complementare, della spettrometria di mobilità ionica intrappolata e della spettrometria di massa a tempo di volo (LC-TIMS-ToF MS/MS) consente la separazione e l'assegnazione PTM delle modifiche biologicamente più rilevanti in un'unica analisi. L'approccio descritto sfrutta i recenti sviluppi nell'acquisizione di dati dipendenti (DDA) utilizzando l'accumulo parallelo nella trappola della mobilità, seguito dalla frammentazione sequenziale e dalla dissociazione indotta dalla collisione. I PTM istonici vengono assegnati con sicurezza in base al tempo di ritenzione, alla mobilità e al modello di frammentazione.

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.

Autore in primo piano: Identificazione migliorata degli isomeri PTM istonici tramite LC-TIMS-ToF MS/MS e PASEF

Screening della modificazione degli istoni mediante cromatografia liquida, spettrometria di mobilità ionica intrappolata e spettrometria di massa a tempo di volo

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.

Un flusso di lavoro proteomico bottom-up (Figura 7) prevede in genere quanto segue: estrazione delle proteine target da un campione grezzo, seguita dalla quantificazione della concentrazione delle proteine e quindi frazionamento, di solito mediante elettroforesi su gel o cromatografia liquida. Dopo il frazionamento, le proteine vengono digerite utilizzando un enzima proteolitico (spesso tripsina) e, infine, l'analisi spettrometrica di massa dei peptidi risultanti e l'identificazione delle pr...

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

Un flusso di lavoro analitico basato sulla cromatografia liquida, sulla spettrometria di mobilità ionica intrappolata e sulla spettrometria di massa a tempo di volo (LC-TIMS-TOF MS/MS) per un'analisi "bottom-up" ad alta affidabilità e altamente riproducibile delle modificazioni istoniche e l'identificazione in base ai parametri principali (tempo di ritenzione [RT], sezione d'urto di collisione [CCS] e rapporto massa-carica [m/z] accurato).

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

Viene proposto un metodo che utilizza un protocollo di estrazione degli istoni con un'efficienza del 95%. Insieme a LC-TIMS-ToF MS/MS, in breve tempo, è possibile ottenere uno screening per quei PTM presenti nell'istone. E soprattutto, la possibilità di separare gli isomeri.La separazione dei peptidi isomerici, sebbene possibile utilizzando la sola spettrometria di massa tandem, è spesso difficile. Incorporando TIMS, introduciamo un'ulteriore dimensione di separazione, semplificando molte identificazioni di isomeri posizionali. Inoltre, la tecnologia passiva aumenta la sensibilità di questi esperimenti, migliorando ulteriormente la nostra capacità di analizzare le variazioni epigenetiche.Con l'aumento della capacità di identificare le posizioni PTM, arriva la capacità di determinare ulteriormente la correlazione tra vari fattori ambientali o biologici e i loro effetti sull'epigenoma. I metodi che migliorano e facilitano l'analisi degli istoni possono essere applicati a diverse aree di ricerca, tra cui la chimica medica e ambientale. La dinamica della conferma delle proteine in stati nativi o danneggiati sarà stabilita dall'implementazione di TIMS MS/MS con HDX.Attraverso LC-TIMS-ToF MS/MS e Nano LC-TIMS-ToF MS/MS, verranno tracciate le differenze di PTM esistenti in vari campioni biologici.

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).