Name: lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics
Uploaded: 2017-04-09T15:00:00
Description: Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

Dit werk werd mede ondersteund door subsidies van de Singapore ministerie van Onderwijs (Tier 2: Grant ARC9 / 15), National Medical Research Council van Singapore (NMRC-OF-IRG-0003-2016) en NTU-NHG Aging Research Grant ( Grant ARG / 14017). We willen graag onze dankbaarheid en meest oprechte dank aan Dr. Andrew Alpert en PolyLC team uit te drukken voor vriendelijk voorzag ons van het verpakkingsmateriaal dat mogelijk deze studie gemaakt.

1. Verpakking de lange lengte Anion exchange (LAX) Capillaire kolom (Noot: Hoewel de LAX kolom kan in-huis verpakt als we in dit protocol beschrijven LOS kolommen zijn ook commercieel verkrijgbaar, zie tabel van Materialen en Reagentia voor verdere details). <ol><li> Suspendeer 50 mg zwakke anionenuitwisselende pakkingsmateriaal in 3,5 ml verpakking buffer (tabel 1) aan de slurrie te bereiden. </li><li> Monteer het uiteinde van de capillaire kolom...

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In deze video-artikel geven we een stap-voor-stap protocol van LERLIC-MS / MS 3, een werkwijze om diepgaande karakterisering voeren en nauwkeurig de mate van proteïne deamidering en enzymatische processen dit eiwitmodificatie. LERLIC-MS / MS is gebaseerd op het gebruik van een lange lengte (50 cm) LOS volgens het elektrostatische repulsie-hilic (Erlić) 27. Het gebruik van een lange-kolomlengte, zoals in onze studie 3, maximaliseert het potentieel ...

Deamidering is een proteïne posttranslationele modificatie (PTM) dat een negatieve lading aan het eiwit skelet geïntroduceerd door middel van modificatie van asparagine (Asn) en / of glutamine (Gln) resten 1. Deze modificatie terwijl beïnvloeden Asn residuen genereert de isomere producten isoaspartaamzuur (isoAsp) en n -aspartic acid (Asp) bij een gemeenschappelijke 3: 1 verhouding 2. Ondanks deze verhouding kan worden gewijzigd door tussenkomst van de reparerende enzym L-isoaspartyl methyltransferase (PIMT) 3, 4. Evenzo deamidering van Gln residuen genereert isomere gamma-glutaminezuur (γ-Glu) en alfa-glutaminezuur isovormen (α-Glu) en een verwachte 1: 7 verhouding van 3, 5, doch deze verhouding kan worden geschoven door de werking van de alomtegenwoordige enzym transglutaminase 2 en andere transglutaminasen, zoals transglutaminase 1, enzyme onlangs geïdentificeerd als geassocieerd met extracellulaire blaasjes in de hersenen 6. De oorsprong van deamidatie kan hetzij spontaan of enzymatisch worden, de eerste is vooral gebruikelijk op Gin residuen waarin transglutaminasen en andere enzymen mediëren inter / intramoleculaire verknoping via transamideringsprodukt (zie 3 voor meer details over Gin transamideringsprodukt en de implicaties verschillende chronische en dodelijke ziekten bij de mens). Daarom, deamidering is een doorvoermechanisme die een cruciale weerslag op de structuur en functie van de betrokken moleculen 4, 7, 8 heeft en vereist een uitgebreide chemische karakterisering 3 in het licht van de diverse biochemische gevolgen inbegrip van de service moleculaire klok van veroudering 9 . Hoewel deamidering Asn residuen is<html> relatief goed gekenmerkt door bottom-up shotgun proteomics 1, 10, deamidatie of Gln-residuen nog steeds geen geschikte karakteriseringsmethode buiten de uitdagende analyse van modelverbindingen door elektronen radicaal fragmentatie 11. We hebben onlangs een nieuwe één dimensie shotgun proteomics strategie (LERLIC-MS / MS) 3 die gescheiden Gin en Asn deamidering isovormen uit complexe biologische monsters en modelverbindingen maakt in een enkele analyse. LERLIC-MS / MS is gebaseerd op de scheiding van de tryptische peptiden gedigesteerd met behulp van een lange lengte (50 cm) ionenuitwisselingskolom (LAX) aan elektrostatische afstoting-hilic (Erlić) stand en gekoppeld aan tandem massaspectrometrie (LC MS / MS). Deze nieuwe analytische strategie is toegepast voor het karakteriseren en kwantificeren betrekkelijk de omvang van elk gedeamideerd residuen in humane hersenweefselsf &quot;&gt; 3. Niettemin zal de hier geschetste protocol videobeelden van LERLIC-MS / MS doel de eigenaardigheden van eiwitdeamiderende bestuderen de biochemische context van belang. ETHISCHE UITSPRAAK Alle procedures van dit protocol zijn goedgekeurd door de Institutional Review Board van de Nanyang Technological University in Singapore en zijn uitgevoerd in overeenstemming met de institutionele richtlijnen.

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			<td height="21" style="height:21px;width:359px;">Protease inhibitor cocktail tablets</td>
			<td style="width:424px;">Roche</td>
			<td style="width:207px;">11836170001 (ROCHE)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Phosphate buffer solution 10X (diluted to 1x)</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">P5493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium acetate</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">A1542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium deoxycholate</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Dithiothreitol</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Iodoacetamide</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">i6125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Formic acid</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">F0507 (HONEYWELL)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ammonium hydroxide</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">338818 (HONEYWELL)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetonitrile HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">675415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Isopropanol HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">675431</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Water HPLC grade</td>
			<td style="width:424px;">Sigma-Aldrich</td>
			<td style="width:207px;">14263</td>
			<td></td>
		</tr>
	</tbody>
</table>

lerlic-ms/ms for in-depth characterization and quantification of glutamine and asparagine deamidation in shotgun proteomics

Karakterisatie van eiwitdeamiderende is noodzakelijk om de rol (len) en de mogelijkheden van deze eiwitten posttranslationele modificatie (PTM) in humane pathologie en andere biochemische context ontcijferen. Ter karakterisering van eiwitten deamidatie voeren, hebben wij onlangs een nieuwe lange-length elektrostatische repulsie-hilic-tandem massaspectrometrie (LERLIC-MS / MS) werkwijze die het glutamine (Gln) en asparagine (Asn) isovorm kan scheiden producten van de deamidatie van modelverbindingen zeer complexe biologische monsters. LERLIC-MS / MS is dus de eerste shotgun proteomics strategie voor de scheiding en kwantificering van Gin deamidatie isovormen. We tonen ook als noviteit, dat het monster verwerkingsprotocol geschetste stabiliseert het succinimide tussenliggende waardoor de karakterisering van LERLIC-MS / MS. Toepassing van LERLIC-MS / MS zoals weergegeven in deze video artikel kan u helpen om op te helderen de op dat moment onbekenden moleculaire arrays van eiwit deamidatie. Bovendien LERLIC-MS / MS verschaft verder inzicht in de enzymatische reacties die deamidatie bij onderscheiden biologische achtergronden omvatten.

Deamidatie of Gin en Asn residu wordt beschouwd als een degeneratieve eiwitmodificatie (DPM) betrokken bij een aantal chronische en dodelijke ziekten 14. Is aangetoond dat dit PTM de halfwaardetijd en afbrekende toestanden van antilichamen en andere moleculen kunnen voorspellen in het menselijk lichaam en soortgelijke biologische backgrounds 1, 15. De betekenis van eiwitdeamiderende, in feite, gaat verder ...

Watch this Scientific Journal Video about LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics at JoVE.com

LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics

Hier geven we een stap-voor-stap protocol van de lange lengte elektrostatische repulsie-hilic-tandem massaspectrometrie (LERLIC-MS / MS) methode. Dit is een nieuwe methode die het mogelijk maakt voor het eerst kwantificering en karakterisering van glutamine en asparagine deamidering isovormen door shotgun proteomics.

LERLIC-MS / MS Gedetailleerde karakterisering en kwantificering van glutamine en asparagine Deamidatie in Shotgun Proteomics

Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in ...

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