Name: Auteur in de schijnwerpers: verbeterde identificatie van histon PTM-isomeren door middel van LC-TIMS-ToF MS/MS en 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

Dit materiaal is gebaseerd op werk dat wordt ondersteund door de National Science Foundation onder subsidienr. HRD-1547798 en subsidienr. HRD-2111661. Deze NSF-beurzen werden toegekend aan de Florida International University als onderdeel van het Centers of Research Excellence in Science and Technology (CREST)-programma. Dit is bijdrage nummer 1672 van het Institute of Environment, een vooraanstaand programma aan de Florida International University. Aanvullende steun werd verleend door het National Institute of Health onder subsidienr. R21AI135469 aan Francisco Fernandez-Lima en Grant No. R01HD106051 aan Benjamin A. Garcia, evenals door de National Science Foundation onder subsidienr. CHE-2127882 aan Benjamin A. Garcia. De auteurs willen graag de aanvankelijke steun van Dr. Mario Gomez Hernandez tijdens de eerste methode-ontwikkelingen erkennen.

OPMERKING: Histon-monsters werden geëxtraheerd met behulp van een methode die is aangepast aan Bhanu et al. (2020)12.
1. Voorbereiding van het monster
<ol><li>Oogsten van gekweekte cellen<ol><li>Wanneer cellen voor 80% samenvloeien, zorg er dan voor dat ze levensvatbaar zijn met behulp van trypanblauwe uitsluiting. OPMERKING: Voor deze experimenten werd een HeLa S3-cellijn gebruikt, maar deze methode kan op alle gekweekte cellen worden toegepast.<...

histon proteomische analyse en karakterisering van posttranslationele modificatie

<ol>
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Histonen zijn basiseiwitten die de chromatinestructuur reguleren door interactie met DNA in de vorm van octameren die bestaan uit de vier kernhistonen (twee H2A, H2B, H3 en H4)20. Histonen bevatten talrijke lysine- en arginineresiduen, die gemakkelijk kunnen worden gemodificeerd, wat leidt tot uitgebreide PTM's die de chromatinechemie veranderen door de histonfunctie te beïnvloeden of door zich te binden aan andere cellulaire eiwitten21. PTM's kunnen biologische reacties u...

In eukaryote cellen wordt DNA verpakt als chromatine in functionele eenheden die nucleosomen worden genoemd. Deze eenheden zijn samengesteld uit een octameer van vier kernhistonen (twee elk van H2A, H2B, H3 en H4)1,2,3,4. Histonen behoren tot de meest voorkomende en best geconserveerde eiwitten in eukaryoten, die grotendeels verantwoordelijk zijn voorgenregulatie5. Histon posttranslationele modificaties (PTM's) spelen een grote rol bij de regulatie van chromatinedynamiek en voeren verschillende biologische processen uit, zoals DNA-transcriptie, replicatie en reparatie6. PTM's komen voornamelijk voor op het toegankelijke oppervlak van de N-terminale gebieden van histonen die in contact staan met DNA 3,7. Staart- en kernmodificaties beïnvloeden echter de chromatinestructuur, veranderen internucleosoominteracties en rekruteren specifieke eiwitten 3,8.
Een huidige uitdaging tijdens proteomics op basis van vloeistofchromatografie-massaspectrometrie (LC-MS) is de potentiële co-elutie van interessante analyten. In het geval van data-afhankelijke analyses (DDA) vertaalt dit zich in het potentiële verlies van verschillende precursorionen tijdens het MS/MS-acquisitieproces9. Time-of-flight (ToF)-instrumenten verwerven spectra met een zeer hoge frequentievan 9,10 (tot tientallen kHz)11; dit maakt ze in staat om snel de totale precursorionen in een complex monster (MS1) te scannen, waardoor ze een optimale gevoeligheid en MS/MS-sequentiesnelheden (tot 100 Hz)9 beloven en ideaal zijn voor biologische monsteranalyse10. Desalniettemin wordt de gevoeligheid die beschikbaar is bij deze hoge scansnelheden beperkt door de MS/MS-snelheid9. De toevoeging van trapped ion mobility spectrometry (TIMS) in combinatie met een orthogonale quadrupole time-of-flight (qToF) massaspectrometer werd gebruikt om deze beperkingen te verminderen. In TIMS worden alle voorloperionen in tandem geaccumuleerd en geëlueerd als functie van hun mobiliteit, in plaats van enkele voorlopermassa's te selecteren met een quadrupool9. Parallelle accumulatie-seriële fragmentatie (PASEF) maakt honderden MS/MS-gebeurtenissen per seconde mogelijk zonder verlies van gevoeligheid9.
Het hoofddoel van dit werk was om de recente ontwikkelingen van DDA te laten zien met behulp van parallelle accumulatie in de mobiliteitsval, gevolgd door sequentiële fragmentatie en botsingsgeïnduceerde dissociatie (CID). Histone PTM's werden met vertrouwen toegewezen op basis van hun retentietijden (RT's), mobiliteit en fragmentatiepatronen.

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			<td>-80 &#176;C Freezer</td>
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			<td>Animal Origin-Free</td>
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			<td>Finntip Flex 1000 &#956;L, nonsterile, nonfiltered, racked tips</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>
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			<td>10x Dulbecco&#8217;s PBS without Ca2+/Mg2+</td>
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			<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>
			<td>Corning</td>
			<td>352070</td>
			<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>
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			<td>P-DW-500-C-S</td>
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			<td>Acetone</td>
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			<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>
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			<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>
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			<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-eiwitten zijn zeer overvloedig aanwezig en geconserveerd onder eukaryoten en spelen een grote rol bij genregulatie als gevolg van structuren die bekend staan als posttranslationele modificaties (PTM's). Door de positie en aard van elke PTM of elk patroon van PTM's te identificeren met betrekking tot externe of genetische factoren, kan deze informatie statistisch worden gecorreleerd met biologische reacties zoals DNA-transcriptie, replicatie of reparatie. In dit werk wordt een high-throughput analytisch protocol beschreven voor de detectie van histon-PTM's uit biologische monsters. Het gebruik van complementaire vloeistofchromatografie, spectrometrie voor de mobiliteit van ingesloten ionen en time-of-flight-massaspectrometrie (LC-TIMS-ToF MS/MS) maakt het mogelijk om de biologisch meest relevante modificaties in één enkele analyse te scheiden en PTM-toe te wijzen. De beschreven aanpak maakt gebruik van recente ontwikkelingen op het gebied van afhankelijke data-acquisitie (DDA) met behulp van parallelle accumulatie in de mobiliteitsval, gevolgd door sequentiële fragmentatie en door botsingen geïnduceerde dissociatie. Histone PTM's worden met vertrouwen toegewezen op basis van hun retentietijd, mobiliteit en fragmentatiepatroon.

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.

Auteur in de schijnwerpers: verbeterde identificatie van histon PTM-isomeren door middel van LC-TIMS-ToF MS/MS en PASEF

Screening op histonmodificatie met behulp van vloeistofchromatografie, Trapped Ion Mobility Spectrometry en Time-Of-Flight Mass Spectrometry

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.

Een bottom-up proteomische workflow (figuur 7) omvat doorgaans het volgende: extractie van het doeleiwit of de doeleiwitten uit een ruw monster, gevolgd door het kwantificeren van de concentratie van het eiwit of de eiwitten, en vervolgens fractionering, meestal door middel van gelelektroforese of vloeistofchromatografie. Na fractionering worden de eiwitten verteerd met behulp van een proteolytisch enzym (vaak trypsine), en ten slotte massaspectrometrische analyse van de resulterende peptide...

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

Een analytische workflow op basis van vloeistofchromatografie, mobiliteitsspectrometrie met ingesloten ionen en time-of-flight-massaspectrometrie (LC-TIMS-ToF MS/MS) voor een zeer betrouwbare en zeer reproduceerbare "bottom-up"-analyse van histonmodificaties en identificatie op basis van de belangrijkste parameters (retentietijd [RT], botsingsdoorsnede [CCS] en nauwkeurige massa-tot-lading [m/z]-verhouding).

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

Er wordt een methode voorgesteld met behulp van het histonextractieprotocol met een efficiëntie van 95%. Samen met LC-TIMS-ToF MS/MS is het mogelijk om in korte tijd een screening te verkrijgen op die PTM's die in het histon aanwezig zijn. En vooral de mogelijkheid om isomeren te scheiden.De scheiding van isomere peptiden is vaak moeilijk met behulp van tandem-massaspectrometrie alleen. Door TIMS op te nemen, introduceren we een extra dimensie van scheiding, waardoor veel positie-isomeeridentificaties worden vereenvoudigd. Bovendien verhoogt passieve technologie de gevoeligheid van deze experimenten, waardoor ons vermogen om epigenetische variaties te analyseren verder wordt verbeterd.Met het toegenomen vermogen om PTM-posities te identificeren, komt er de mogelijkheid om de correlatie tussen verschillende omgevings- of biologische factoren en hun effecten op het epigenoom verder te bepalen. Methoden die de analyse van histonen verbeteren en vergemakkelijken, kunnen worden toegepast op verschillende onderzoeksgebieden, waaronder medische en milieuchemie. De dynamiek van eiwitbevestiging in inheemse of beschadigde toestanden zal worden vastgesteld op basis van de implementatie van TIMS MS/MS met HDX.Door middel van LC-TIMS-ToF MS/MS en Nano LC-TIMS-ToF MS/MS zullen de verschillen in PTM in verschillende biologische monsters in kaart worden gebracht.

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).