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

Dette arbejde blev delvist støttet af tilskud fra Singapore Undervisningsministeriet (Tier 2: Grant ARC9 / 15), National Council of Singapore (NMRC-AF-IRG-0003-2016), og NTU-NHG Aldringsforskning Grant Medical Research ( Grant ARG / 14017). Vi vil gerne udtrykke vores taknemmelighed og mest oprigtige tak til Dr. Andrew Alpert og PolyLC team for venligt forsynet os med den emballage, den muliggjorde denne undersøgelse.

1. Pakning af lange længde Anion-bytning (LAX) kapillarsøjle (Bemærk: Selv om LAX kolonne kan være i hjemmet pakket som vi beskriver i denne protokol, LAX kolonner er også kommercielt tilgængelige, se tabel Materialer og reagenser for yderligere detaljer). <ol><li> Suspendere 50 mg svag anionbytter pakkemateriale i 3,5 ml pakning puffer (tabel 1) til fremstilling af opslæmningen. </li><li> Samle enden af ​​søjlen (50 cm længde - 200 u...

<ol>
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I denne video-artikel præsenterer vi en trin-for-trin-protokollen af LERLIC-MS / MS 3, en fremgangsmåde udføre grundig karakterisering og til nøjagtigt at bestemme omfanget af protein deamidering og enzymatiske processer, der er involveret på dette protein modifikation. LERLIC-MS / MS er baseret på anvendelsen af en lang længde (50 cm) LAX henhold til princippet om elektrostatisk frastødning-hydrofil interaktionskromatografi (ERLIC) 27. Anvendelsen af en lang læng...

Deamidering er et protein posttranslationel modifikation (PTM), som indfører en negativ ladning til proteinet backbone ved modifikation af asparagin (Asn) og / eller glutamin (Gin) resterne 1. Denne modifikation mens påvirke Asn-rester genererer isomere produkter isoaspartic syre (isoAsp) og n asparaginsyre (Asp) ved en fælles 3: 1 forhold 2. Uanset, kan dette forhold ændres ved indgriben fra reparation enzym L-isoaspartyl methyltransferase (PIMT) 3, 4. Tilsvarende deamidering af Gin rester genererer den isomere gamma-glutaminsyre (γ-Glu) og alfa-glutaminsyre isoformer (α-Glu) på et forventet 1: 7 forhold 3, 5, men dette forhold kan forskydes ved indvirkning af den allestedsnærværende enzym transglutaminase 2 og andre transglutaminaser, herunder transglutaminase 1, en enzyme nylig identificeret som er forbundet med ekstracellulære vesikler i hjernen 6. Oprindelsen af deamidering kan enten spontan eller enzymatisk, førstnævnte er især almindeligt på Gin-rester, hvori transglutaminaser og andre enzymer medierer inter / intra-molekylær tværbinding via transamidering (se 3 for yderligere detaljer om Gin transamidering og dens konsekvenser i flere kronisk og dødelige sygdomme hos mennesker). Derfor, deamidering er en PTM, der har en afgørende indflydelse på den struktur og funktion af berørte molekyler 4, 7, 8 og kræver en tilbundsgående kemiske karakterisering 3 i lyset af dets forskellige biokemiske konsekvenser herunder dets tjeneste som molekylære ur af ældning 9 . Skønt deamidering af Asn-rester er blevet<html> relativt velkarakteriseret ved bottom-up shotgun proteomics 1, 10, deamidering af Gin rester stadig ikke har en egnet karakterisering fremgangsmåde ud over den udfordrende analyse af modelforbindelser ved elektron-radikal fragmentering 11. Vi har for nylig udviklet en ny én dimension shotgun proteomics strategi (LERLIC-MS / MS) 3, der muliggør adskillelse af Gin og Asn deamideringssteder isoformer fra komplekse biologiske prøver og modelforbindelser i en enkelt analyse. LERLIC-MS / MS er baseret på adskillelsen af ​​tryptiske spaltet peptider under anvendelse af en lang længde (50 cm) ionbyttersøjle (LAX) arbejder på elektrostatisk frastødning-hydrofil interaktionskromatografi (ERLIC) -tilstand og koblet til tandem-massespektrometri (LC- MS / MS). Denne nye analytiske strategi er blevet anvendt til at karakterisere og relativt kvantificere udstrækningen af ​​de deamideret rest i humane hjernevævf &quot;&gt; 3. Ikke desto mindre vil protokollen beskrevet her giver video billeddannelse af LERLIC-MS / MS til formål at studere de særlige forhold protein deamidering i den biokemiske sammenhæng af interesse. ETIK OVERSIGT Alle procedurer vedrørende denne protokol er blevet godkendt af den institutionelle gennemgang bestyrelsen for Nanyang Technological University i Singapore og er udført i overensstemmelse med de institutionelle retningslinier.

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			<td height="21" style="height:21px;width:359px;">Sequencing-grade modified trypsin</td>
			<td style="width:424px;">Promega</td>
			<td style="width:207px;">V5111</td>
			<td></td>
		</tr>
		<tr height="21">
			<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

Karakterisering af protein deamidering er bydende nødvendigt at dechifrere den rolle (r) og muligheder af dette protein posttranslationel modifikation (PTM) i human patologi og andre biokemiske sammenhænge. For at udføre karakterisering af protein deamidering, har vi for nylig udviklet en ny lang længde elektrostatisk frastødning-hydrofil interaktionskromatografi-tandem massespektrometri (LERLIC-MS / MS) metode, der kan adskille glutamin (Gln) og asparagin (Asn) isoform produkter af deamidering fra modelforbindelser til meget komplekse biologiske prøver. LERLIC-MS / MS er derfor den første shotgun proteomics strategi til separation og kvantificering af Gin deamideringssteder isoformer. Vi demonstrerer også, som en nyhed, at prøven behandlingsprotokol her skitserede stabiliserer succinimid mellemprodukt tillader dets karakterisering ved LERLIC-MS / MS. Anvendelse af LERLIC-MS / MS, som vist i denne video artikel kan hjælpe til at belyse den aktuelt unknown molekylære opstillinger af protein deamidering. Derudover LERLIC-MS / MS tilvejebringer yderligere forståelse af de enzymatiske reaktioner, der omfatter deamidering i distinkte biologiske baggrunde.

Deamidering af Gin og Asn-rester betragtes som en degenerativ proteinmodifikation (DPM) impliceret i flere kroniske og dødelige sygdomme 14. Det er blevet påvist, at denne PTM kan forudsige halveringstid og nedbrydende tilstande af antistoffer og andre molekyler i menneskekroppen og lignende biologiske baggrunde 1, 15. Betydningen af protein deamidering, i virkeligheden, går ud over den biomedicinske ko...

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

Her præsenterer vi en trin-for-trin-protokollen af ​​den lange længde elektrostatisk frastødning-hydrofil interaktionskromatografi-tandem massespektrometri (LERLIC-MS / MS) metode. Dette er en hidtil ukendt metode, som gør det muligt for første gang kvantificering og karakterisering af glutamin og asparagin deamideringssteder isoformer med shotgun proteomics.

LERLIC-MS / MS for Indgående Karakterisering og Kvantificering af glutamin og asparagin deamidering i 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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