Name: deep proteome profiling by isobaric labeling, extensive liquid chromatography, mass spectrometry, and software-assisted quantification
Uploaded: 2017-11-15T13:00:00
Description: Watch this Scientific Journal Video about Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification at JoVE.com

De auteurs bedanken alle andere lab en faciliteit leden voor nuttige discussie. Dit werk werd slechts gedeeltelijk ondersteund door NI H verleent R01GM114260, R01AG047928, R01AG053987 en ALSAC. De MS-analyse werd uitgevoerd in de St. Jude Children's Research Hospital Proteomics Facility, slechts gedeeltelijk ondersteund door de NIH Cancer Center ondersteuning subsidie P30CA021765. De auteurs bedanken Nisha Badders voor hulp bij het bewerken van het manuscript.

Let op: Raadpleeg alle relevante gegevens omtrent de veiligheid (d.w.z., MSDS) vóór gebruik. Gebruik bij het uitvoeren van dit protocol op alle passende veiligheidspraktijken.
 Opmerking: een TMT 10-plex isobaar label reagens set wordt gebruikt in dit protocol voor het proteoom kwantificatie van 10 monsters.
 1. voorbereiding van de cellen/weefsels Opmerking: het is van cruciaal belang voor het verzamelen van monsters in minima...

<ol>
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Beschrijven we een hoge-doorvoer-protocol voor de kwantificatie van eiwitten met een 10-plex isobaar labeling strategie, die is met succes geïmplementeerd in verschillende publicaties12,13,14,32 . In dit protocol, kunnen we maximaal 10 verschillende biologische EiwitSteekproeven in 1 experiment analyseren. We kunnen routinematig identificeren en kwantificeren van ruim 10.000 eiwitten met hoge ...

Vooruitgang in de technologie van de volgende generatie sequencing hebben geleid tot een nieuw landschap voor de studie van biologische systemen en ziekten bij de mens. Dit heeft een groot aantal metingen van het genoom, transcriptome Proteoom, metabolome en andere moleculaire systemen tot tastbare toegestaan. De Spectrometrie van de massa (MS) is een van de meest gevoelige methoden in de analytische chemie, en de toepassing ervan in proteomics is snel uitgebreid na de sequencing van het menselijk genoom. Op het gebied van proteomics, hebben de afgelopen jaren grote vooruitgang in op basis van MS kwantitatieve analyses, met inbegrip van isobaar labeling en multiplexing vermogen gecombineerd met uitgebreide vloeistofchromatografie, naast instrumentatie opgeleverd vooruitgang, waardoor voor snellere, meer nauwkeurige metingen met minder monstermateriaal nodig. Kwantitatieve proteomics geworden een reguliere aanpak voor profilering van tienduizenden van eiwitten en posttranslationele modificaties in zeer complexe biologische monsters1,,2,,3,4 , 5 , 6.
Multiplexed isobaar labeling methoden zoals isobaar tag voor relatieve en absolute kwantificatie (dat wil zeggen, iTRAQ) en tandem massa tag (TMT) MS hebben sterk verbeterd monsters worden verwerkt en steeg het aantal monsters dat kan worden geanalyseerd in één 1,6,7,8te experimenteren. Samen met andere kwantificatie op basis van MS-methoden, zoals label-vrije kwantificatie en stabiele isotoop labelen met aminozuren in de cultuur van de cel (dat wil zeggen, SILAC), het potentieel van deze technieken in de proteomics is veld aanzienlijke9 ,10,11. De TMT-methode kunnen bijvoorbeeld 10 EiwitSteekproeven te samen in 1 experiment worden geanalyseerd met behulp van 10-plex reagentia. Deze structureel identiek TMT-tags hebben dezelfde totale massa, maar zware isotopen zijn differentieel verdeeld over kooldioxide of stikstof-atomen, resulterend in een unieke verslaggever ion tijdens MS/MS versnippering van elke tag, waardoor de relatieve kwantificatie tussen de 10 monsters. De strategie van de TMT is routinematig toegepast om te bestuderen van de biologische afbraak, progressie van de ziekte, en cellulaire processen12,13,14.
Aanzienlijke technische verbeteringen hebben vloeistofchromatografie (LC)-MS/MS systemen, zowel in termen van LC scheidingen en MS parameters, eiwit identificatie maximaliseren zonder in te boeten kwantificatie nauwkeurigheid verbeterd. Eerste-dimensie scheiding voor peptides door een scheiding techniek met hoge orthogonaliteit aan de tweede dimensie is van cruciaal belang is in dit soort geweer proteomics methode om maximale resultaten20. Hoge-pH reversed-phase vloeistofchromatografie (RPLC) levert betere prestaties dan conventionele sterke catie-uitwisseling chromatografie20. Als hoge-pH RPLC wordt gecombineerd met een tweede dimensie van lage-pH RPLC, zowel analytische dynamisch bereik en eiwit dekking zijn verbeterd, resulterend in de mogelijkheid om het grootste deel van de geuite eiwitten te identificeren bij het uitvoeren van geheel-Proteoom analyse15 ,16,17,18. Andere technische ontwikkelingen omvatten kleine C18 deeltjes (1.9 µm) en uitgebreid lang kolom (~ 1 m)19. Bovendien omvatten andere opmerkelijke verbeteringen nieuwe versies van massaspectrometers met snelle scan tarieven, verbeterde gevoeligheid en resolutie20en verfijnde bioinformatics pijpleidingen voor MS gegevens mijnbouw21.
Hier beschrijven we een gedetailleerd protocol waarin de meest recente methodologieën met wijzigingen ter verbetering van zowel de gevoeligheid en de doorvoer, met de nadruk op kwaliteit controlemechanismen gedurende het gehele experiment. Het protocol bevat eiwit extractie en spijsvertering, TMT 10-plex labeling fundamentele pH en zure pH RPLC fractionering, high-resolution MS detectie en verwerking van de gegevens van de MS (Figuur 1). Bovendien voeren we verschillende kwaliteitscontrole stappen voor het oplossen van problemen en de evaluatie van de experimentele variatie. Dit gedetailleerde protocol is bedoeld om onderzoekers te helpen nieuwe naar het veld routinematig identificeren en duizenden eiwitten uit een lysate nauwkeurig te kwantificeren of weefsel.

<table>
	<tbody>
		<tr>
			<td>1220 LC system</td>
			<td>Agilent</td>
			<td>G4288B</td>
			<td></td>
		</tr>
		<tr>
			<td>50% Hydroxylamine</td>
			<td>Thermo Scientific</td>
			<td>90115</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH015-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bullet Blender</td>
			<td>Next Advance</td>
			<td>BB24-AU</td>
			<td></td>
		</tr>
		<tr>
			<td>Butterfly Portfolio Heater</td>
			<td>Phoenix S&#38;T</td>
			<td>PST-BPH-20</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 tips</td>
			<td>Harvard Apparatus</td>
			<td>74-4607</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>41648</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector</td>
			<td>Gilson</td>
			<td>FC203B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>Next Advance</td>
			<td>GB05</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma</td>
			<td>I6125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lys-C</td>
			<td>Wako</td>
			<td>125-05061</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Burdick &#38; Jackson</td>
			<td>AH230-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>Q Exactive HF</td>
			<td></td>
		</tr>
		<tr>
			<td>nanoflow UPLC</td>
			<td>Thermo Scientific</td>
			<td>Ultimate 3000</td>
			<td></td>
		</tr>
		<tr>
			<td>ReproSil-Pur C18 resin, 1.9um</td>
			<td>Dr. Maisch GmbH</td>
			<td>r119.aq.0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Self Pck Columns</td>
			<td>New Objective</td>
			<td>PF360-75-15-N-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>Speedva</td>
			<td>Thermo Scientific</td>
			<td>SPD11V</td>
			<td></td>
		</tr>
		<tr>
			<td>TMT 10plex Isobaric label reagent</td>
			<td>Thermo Scientific</td>
			<td>90110</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Applied Biosystems</td>
			<td>400003</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V511C</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Xbridge Column C18 column</td>
			<td>Waters</td>
			<td>186003943</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziptips C18</td>
			<td>Millipore</td>
			<td>ZTC18S096</td>
			<td></td>
		</tr>
		<tr>
			<td>SepPak 1cc 50mg</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td></td>
		</tr>
	</tbody>
</table>

deep proteome profiling by isobaric labeling, extensive liquid chromatography, mass spectrometry, and software-assisted quantification

Vele uitzonderlijke vooruitgang geboekt in de Spectrometrie van de massa (MS)-op basis van proteomics, met bijzondere technische vooruitgang in vloeistofchromatografie (LC) gekoppeld aan massaspectrometrie tandem (LC-MS/MS) en isobaar labeling multiplexing capaciteit. Hier introduceren we een diep-proteomics profiling protocol dat 10-plex tandem massa tag (TMT combineert) labelen met een uitgebreide LC/LC-MS/MS platform, en na MS computationele interferentie correctie op het hele proteomes nauwkeurig te kwantificeren. Dit protocol omvat de volgende hoofdstappen: eiwit extractie en spijsvertering, labelen van de TMT, 2-dimensionale (2D) LC, hoge-resolutie massa spectrometrie en computationele gegevensverwerking. Kwaliteitscontrole stappen zijn opgenomen voor het oplossen van problemen en de evaluatie van de experimentele variatie. Meer dan 10.000 eiwitten in zoogdieren monsters kunnen vol vertrouwen met dit protocol worden quantitated. Dit protocol kan ook worden toegepast op de kwantificatie van post-vertalende wijzigingen met kleine wijzigingen. Deze methode multiplexed, robuuste biedt een krachtige tool voor proteoom analyse in een verscheidenheid van complexe monsters, met inbegrip van celkweek, dierlijke weefsels en menselijke klinische specimens.

We gebruikten een eerder beschreven Kruis-soorten peptide mix om systematisch analyseren het effect van de compressie verhouding in 3 stappen van de grote protocol, met inbegrip van pre-MS fractionering, MS-instellingen en post-MS correctie23. De pre-MS fractionering werd geëvalueerd en geoptimaliseerd met behulp van een combinatie van fundamentele pH RPLC en zure pH RPLC. Voor de analyse van de post-MS, werden alleen soortspecifieke peptiden beschouwd. We gebruik...

Watch this Scientific Journal Video about Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification at JoVE.com

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

We presenteren een protocol om te kwantificeren nauwkeurig eiwitten met isobaar etikettering, uitgebreide fractionering, bioinformatica tools en kwaliteitscontrole stappen in combinatie met vloeibare chromatografie geïnterfacet met een hoge resolutie massaspectrometer.

Diepe Proteoom profilering door isobaar Labeling, uitgebreide vloeistofchromatografie, massaspectrometrie en Software-bijgewoonde kwantificering

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Research

JoVE Journal

Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless capillaire elektroforese-massaspectrometrie voor metabole of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Tijdsgeresolveerde elektrosprayionisatie waterstof-deuterium Exchange Massaspectrometrie voor het bestuderen van Protein Structure en Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Het oplossen van Affiniteit gezuiverde eiwit complexen door Blue inheemse PAGE en Protein Correlatie Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

Een vloeistofchromatografie tandem-massaspectrometrie gebaseerde benadering voor metabolietanalyse van<em&gt; Staphylococcus aureus</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Detectie van eiwitubiquitinatiesites door peptideverrijking en massaspectrometrie

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Proteoom bestrijkende kwantificering van Labeling homogeniteit op het niveau van één molecuul

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptide profilering en identificatie geïllustreerd door angiotensine II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Absolute kwantificering van de Celvrije eiwitsynthese metabolisme door omgekeerde fase vloeistofchromatografie-massaspectrometrie

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Label-vrije Immunoprecipitation massaspectrometrie workflow voor grootschalige nucleaire interactome profilering

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Kwantitatieve analyse van de cellulaire lipidoom van Saccharomyces Cerevisiae met behulp van vloeibare chromatografie in combinatie met tandem massaspectrometrie

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...