Name: Författare i fokus: Förbättrad identifiering av PTM-isomer med histontoner genom LC-TIMS-ToF MS/MS och 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

Detta material är baserat på arbete som stöds av National Science Foundation under Grant No. HRD-1547798 och anslagsnummer. HRD-2111661. Dessa NSF-bidrag tilldelades Florida International University som en del av programmet Centers of Research Excellence in Science and Technology (CREST). Detta är bidrag nummer 1672 från Institute of Environment, ett framstående program vid Florida International University. Ytterligare stöd tillhandahölls av National Institute of Health inom ramen för anslag nr. R21AI135469 till Francisco Fernandez-Lima och Grant No. R01HD106051 till Benjamin A. Garcia, samt av National Science Foundation under anslagsnummer. CHE-2127882 till Benjamin A. Garcia. Författarna vill tacka Dr. Mario Gomez Hernandez för det initiala stödet under den inledande metodutvecklingen.

OBS: Histonprover extraherades med en metod anpassad från Bhanu et al. (2020)12.
1. Beredning av prover
<ol><li>Skörd av odlade celler<ol><li>När cellerna är 80 % sammanflytande, se till att de är livskraftiga med hjälp av trypanblå uteslutning. OBS: En HeLa S3-cellinje användes för dessa experiment, men denna metod kan tillämpas på alla odlade celler.</li>
		<li>Aspirera mediet och applicera sedan 5 ml 1x fosfatbuffrad koksaltlösning (...

Histonproteomikanalys och karakterisering av posttranslationell modifiering

<ol>
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Hitoner är basiska proteiner som reglerar kromatinstrukturen genom att interagera med DNA i form av oktamerer som består av de fyra kärnhistonerna (två vardera av H2A, H2B, H3 och H4)20. Hitoner innehåller många lysin- och argininrester, som lätt modifieras, vilket leder till omfattande PTM som förändrar kromatinkemin genom att påverka histonfunktionen eller genom att binda till andra cellulära proteiner21. PTM kan framkalla biologiska reaktioner genom att arbeta...

I eukaryota celler förpackas DNA som kromatin i funktionella enheter som kallas nukleosomer. Dessa enheter består av en oktamer av fyra kärnhistoner (två vardera av H2A, H2B, H3 och H4)1,2,3,4. Histoner är bland de vanligaste och mest bevarade proteinerna i eukaryoter, som till stor del är ansvariga för genreglering5. Histonposttranslationella modifieringar (PTM) spelar en stor roll i regleringen av kromatindynamiken och riggar olika biologiska processer såsom DNA-transkription, replikation och reparation6. PTM förekommer främst på den tillgängliga ytan av de N-terminala regionerna av histoner som är i kontakt med DNA 3,7. Modifieringar av svans och kärna påverkar dock kromatinstrukturen, förändrar interaktioner mellan nukleosomer och rekryterar specifika proteiner 3,8.
En aktuell utmaning inom vätskekromatografi-masspektrometri (LC-MS)-baserad proteomik är den potentiella samelueringen av analyter av intresse. När det gäller databeroende analyser innebär detta en potentiell förlust av flera prekursorjoner under förvärvsprocessen mellan medlemsstaterna och medlemsstaterna9. Flygtidsinstrument (ToF) samlar in spektra vid mycket hög frekvens 9,10 (upp till tiotals kHz)11. Detta gör dem kapabla att snabbt skanna de totala prekursorjonerna i ett komplext prov (MS1), vilket lovar optimal känslighet och MS/MS-sekvenseringshastigheter (upp till 100 Hz)9 och gör dem idealiska för biologisk provanalys10. Den känslighet som är tillgänglig vid dessa höga skanningshastigheter begränsas dock av MS/MS-frekvensen9. Tillägget av TIMS (Trapped Ion Mobility Spectrometry) i kombination med en ortogonal kvadrupol time-of-flight (qToF) masspektrometer användes för att mildra dessa begränsningar. I TIMS ackumuleras alla prekursorjoner i tandem och elueras som en funktion av deras rörlighet, snarare än att välja enskilda prekursormassor med en kvadrupol9. Parallell ackumulering-seriell fragmentering (PASEF) möjliggör hundratals MS/MS-händelser per sekund utan förlust av känslighet9.
Huvudsyftet med detta arbete var att visa den senaste utvecklingen av DDA med hjälp av parallell ackumulering i mobilitetsfällan följt av sekventiell fragmentering och kollisionsinducerad dissociation (CID). Histone PTM:er tilldelades med säkerhet baserat på deras retentionstider (RT), mobilitet och fragmenteringsmönster.

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			<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

Histonproteiner är mycket vanliga och bevarade bland eukaryoter och spelar en stor roll i genreglering som ett resultat av strukturer som kallas posttranslationella modifieringar (PTM). Genom att identifiera positionen och arten av varje PTM eller mönster av PTM med hänvisning till externa eller genetiska faktorer kan denna information vara statistiskt korrelerad med biologiska svar såsom DNA-transkription, replikation eller reparation. I detta arbete beskrivs ett analysprotokoll med hög kapacitet för detektion av histon-PTM från biologiska prover. Användningen av komplementär vätskekromatografi, spektrometri med fångad jonrörlighet och time-of-flight-masspektrometri (LC-TIMS-ToF MS/MS) möjliggör separation och PTM-tilldelning av de mest biologiskt relevanta modifieringarna i en enda analys. Det beskrivna tillvägagångssättet drar nytta av den senaste utvecklingen inom beroende datainsamling (DDA) med hjälp av parallell ackumulering i mobilitetsfällan, följt av sekventiell fragmentering och kollisionsinducerad dissociation. Histone PTM:er tilldelas med säkerhet baserat på deras retentionstid, rörlighet och fragmenteringsmönster.

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.

Författare i fokus: Förbättrad identifiering av PTM-isomer med histontoner genom LC-TIMS-ToF MS/MS och PASEF

Screening av histonmodifiering med vätskekromatografi, spektrometri för fångad jonrörlighet och masspektrometri för flygtid

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.

Ett proteomikarbetsflöde nedifrån och upp (figur 7) omfattar vanligtvis följande: extraktion av målproteinet eller målproteinerna från ett råprov, följt av kvantifiering av koncentrationen av proteinet/proteinerna och sedan fraktionering, vanligtvis genom gelelektrofores eller vätskekromatografi. Efter fraktionering bryts proteinerna med hjälp av ett proteolytiskt enzym (ofta trypsin), och slutligen masspektrometrisk analys av de resulterande peptiderna och proteinidentifiering med...

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

Ett analytiskt arbetsflöde baserat på vätskekromatografi, spektrometri för fångad jonrörlighet och time-of-flight-masspektrometri (LC-TIMS-ToF MS/MS) för hög konfidens och mycket reproducerbar "bottom-up"-analys av histonmodifieringar och identifiering baserat på huvudparametrar (retentionstid [RT], kollisionstvärsnitt [CCS] och exakt massa-till-laddningsförhållande [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 ...

En metod föreslås med hjälp av histonextraktionsprotokoll med 95% effektivitet. Tillsammans med LC-TIMS-ToF MS/MS är det på kort tid möjligt att få en screening för de PTM som finns i histonen. Och framför allt möjligheten att separera isomerer.Separationen av isomera peptider är ofta svår med enbart tandemmasspektrometri. Genom att införliva TIMS introducerar vi en extra dimension av separation, vilket förenklar många positionsisomeridentifieringar. Dessutom ökar passiv teknik känsligheten i dessa experiment, vilket ytterligare förbättrar vår förmåga att analysera epigenetiska variationer.Med den ökade förmågan att identifiera PTM-positioner kommer möjligheten att ytterligare bestämma sambandet mellan olika miljömässiga eller biologiska faktorer, och deras effekter på epigenomet. Metoder som förbättrar och underlättar analys av histoner kan tillämpas inom flera forskningsområden, bland annat medicinsk kemi och miljökemi. Dynamiken för proteinbekräftelse i naturligt eller skadat tillstånd kommer att fastställas från implementeringen av TIMS MS/MS med HDX.Genom LC-TIMS-ToF MS/MS och Nano LC-TIMS-ToF MS/MS kommer skillnaderna i PTM som finns i olika biologiska prover att kartläggas.

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).