Este material se basa en el trabajo apoyado por la Fundación Nacional de Ciencias bajo la Subvención No. HRD-1547798 y la Subvención No. HRD-2111661. Estas subvenciones de la NSF fueron otorgadas a la Universidad Internacional de Florida como parte del Programa de los Centros de Excelencia en Investigación en Ciencia y Tecnología (CREST). Esta es la contribución número 1672 del Instituto de Medio Ambiente, un programa preeminente de la Universidad Internacional de Florida. El Instituto Nacional de Salud prestó apoyo adicional en el marco de la subvención No. R21AI135469 a Francisco Fernández-Lima y a la Subvención No. R01HD106051 a Benjamín A. García, así como por la Fundación Nacional de Ciencias bajo la Subvención No. CHE-2127882 a Benjamín A. García. Los autores desean agradecer el apoyo inicial del Dr. Mario Gómez Hernández durante el desarrollo inicial del método.

Análisis proteómico de histonas y caracterización de modificaciones postraduccionales

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Melvin A. Park y Matthew Willetts son empleados de Bruker Daltonics Inc., el fabricante del instrumento timsTOF.

Las histonas son proteínas básicas que regulan la estructura de la cromatina interactuando con el ADN en forma de octámeros formados por las cuatro histonas centrales (dos de H2A, H2B, H3 y H4)20. Las histonas contienen numerosos residuos de lisina y arginina, que se modifican fácilmente, lo que da lugar a extensos PTM que alteran la química de la cromatina al influir en la función de las histonas o al unirse a otras proteínas celulares21. Los PTM pueden provocar res...

En las células eucariotas, el ADN se empaqueta como cromatina en unidades funcionales llamadas nucleosomas. Estas unidades están compuestas por un octámero de cuatro histonas centrales (dos de H2A, H2B, H3 y H4)1,2,3,4. Las histonas se encuentran entre las proteínas más abundantes y mejor conservadas en los eucariotas, que son en gran parte responsables de la regulación génica5. Las modificaciones postraduccionales de histonas (PTM) desempeñan un papel importante en la regulación de la dinámica de la cromatina y en la preparación de diversos procesos biológicos, como la transcripción, replicacióny reparación del ADN. Los PTM se encuentran principalmente en la superficie accesible de las regiones N-terminales de las histonas que están en contacto con el ADN 3,7. Sin embargo, las modificaciones de la cola y el núcleo influyen en la estructura de la cromatina, alterando las interacciones entre nucleosomas y reclutando proteínas específicas 3,8.
Un reto actual durante la proteómica basada en cromatografía líquida y espectrometría de masas (LC-MS) es la posible coelución de analitos de interés. En el caso de los análisis dependientes de los datos (DDA), esto se traduce en la pérdida potencial de varios iones precursores durante el proceso de adquisición de MS/MS9. Los instrumentos de tiempo de vuelo (ToF) adquieren espectros a muy alta frecuencia 9,10 (hasta decenas de kHz)11; esto los hace capaces de escanear rápidamente el total de iones precursores dentro de una muestra compleja (MS1), lo que promete una sensibilidad óptima y velocidades de secuenciación MS/MS (hasta 100 Hz)9 y los hace ideales para el análisis de muestras biológicas10. Sin embargo, la sensibilidad disponible a estas altas velocidades de exploración está limitada por la velocidad MS/MS9. Para mitigar estas limitaciones, se utilizó la adición de espectrometría de movilidad de iones atrapados (TIMS) en combinación con un espectrómetro de masas de tiempo de vuelo cuadrupolar ortogonal (qToF). En TIMS, todos los iones precursores se acumulan en tándem y se eluyen en función de su movilidad, en lugar de seleccionar masas precursoras individuales con un cuadrupolo9. La fragmentación en serie de acumulación paralela (PASEF) permite cientos de eventos MS/MS por segundo sin pérdida de sensibilidad9.
El objetivo principal de este trabajo fue mostrar los desarrollos recientes de DDA utilizando la acumulación paralela en la trampa de movilidad seguida de fragmentación secuencial y disociación inducida por colisión (CID). Los PTM de Histone se asignaron con confianza en función de sus tiempos de retención (RT), movilidades y patrones de fragmentación.

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			<td>-80 &#176;C Freezer</td>
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			<td>Falcon Conical Centrifuge Tubes</td>
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			<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>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>179124</td>
			<td>ACS reagent, &#8805;99.5%</td>
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			<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>
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			<td>Bruker Daltonics</td>
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			<td>Compass IsotopePattern</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Compass timsControl 4.1</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
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		<tr>
			<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>
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			<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>
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			<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

Las proteínas histonas son muy abundantes y se conservan entre los eucariotas y desempeñan un papel importante en la regulación génica como resultado de estructuras conocidas como modificaciones postraduccionales (PTM). La identificación de la posición y la naturaleza de cada PTM o patrón de PTM en referencia a factores externos o genéticos permite correlacionar estadísticamente esta información con respuestas biológicas como la transcripción, replicación o reparación del ADN. En el presente trabajo se describe un protocolo analítico de alto rendimiento para la detección de histonas PTM a partir de muestras biológicas. El uso de cromatografía líquida complementaria, espectrometría de movilidad de iones atrapados y espectrometría de masas de tiempo de vuelo (LC-TIMS-ToF MS/MS) permite la separación y la asignación de PTM de las modificaciones biológicamente más relevantes en un solo análisis. El enfoque descrito aprovecha los desarrollos recientes en la adquisición de datos dependientes (DDA) utilizando la acumulación paralela en la trampa de movilidad, seguida de la fragmentación secuencial y la disociación inducida por colisiones. Los PTM de Histone se asignan con confianza en función de su tiempo de retención, movilidad y patrón de fragmentación.

In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)1,2,3,4. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation5. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair6. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA3,7. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins3,8.
A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process9. Time-of-flight (ToF) instruments acquire spectra at very high frequency9,10 (up to tens of kHz)11; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)9 and making them ideal for biological sample analysis10. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate9. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole9. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity9.
The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.

Autor destacado: Identificación mejorada de isómeros PTM de histonas a través de LC-TIMS-ToF MS/MS y PASEF

Cribado de modificación de histonas mediante cromatografía líquida, espectrometría de movilidad de iones atrapados y espectrometría de masas de tiempo de vuelo

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 flujo de trabajo proteómico ascendente (Figura 7) suele implicar lo siguiente: extracción de la(s) proteína(s) objetivo(s) de una muestra cruda, seguida de la cuantificación de la(s) concentración(es) de la(s) proteína(s), y luego fraccionamiento, generalmente mediante electroforesis en gel o cromatografía líquida. Después del fraccionamiento, las proteínas se digieren utilizando una enzima proteolítica (a menudo tripsina) y, finalmente, el análisis por espectrometría de masa...

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 flujo de trabajo analítico basado en cromatografía líquida, espectrometría de movilidad de iones atrapados y espectrometría de masas de tiempo de vuelo (LC-TIMS-ToF MS/MS) para un análisis "ascendente" de alta confianza y altamente reproducible de las modificaciones e identificación de histonas en función de los parámetros principales (tiempo de retención [RT], sección transversal de colisión [CCS] y relación masa-carga [m/z] precisa).

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

Se propone un método que utiliza un protocolo de extracción de histonas con una eficiencia del 95%. Junto con LC-TIMS-ToF MS/MS, en poco tiempo, es posible obtener un cribado de aquellos PTM presentes en la histona. Y sobre todo, la posibilidad de separar isómeros.La separación de péptidos isoméricos, aunque es posible utilizando solo la espectrometría de masas en tándem, suele ser difícil. Al incorporar TIMS, introducimos una dimensión adicional de separación, simplificando muchas identificaciones de isómeros posicionales. Además, la tecnología pasiva aumenta la sensibilidad de estos experimentos, mejorando aún más nuestra capacidad para analizar variaciones epigenéticas.Con la mayor capacidad de identificar las posiciones de PTM, viene la capacidad de determinar aún más la correlación entre varios factores ambientales o biológicos y sus efectos en el epigenoma. Los métodos que mejoran y facilitan el análisis de las histonas se pueden aplicar a varias áreas de investigación, incluida la química médica y ambiental. La dinámica de la confirmación de proteínas en estados nativos o dañados se establecerá a partir de la implementación de TIMS MS/MS con HDX.A través de LC-TIMS-ToF MS/MS y Nano LC-TIMS-ToF MS/MS, se graficarán las diferencias en PTM existentes en varias muestras biológicas.

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

783 vistas.  Florida International University. Se propone un método que utiliza un protocolo de extracción de histonas con una eficiencia del 95%. Junto con LC-TIMS-ToF MS/MS, en poco tiempo, es posible obtener un cribado de aquellos PTM presentes en la histona. Y sobre todo, la posibilidad de separar isómeros.La separación de péptidos isoméricos, aunque es posible utilizando solo la espectrometría de masas en tándem, suele ser difícil. Al incorporar TIMS, introducimos una dimensión adicional de separación, simplificando...

Video: Autor destacado: Identificación mejorada de isómeros PTM de histonas a través de LC-TIMS-ToF MS/MS y PASEF

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).