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

Detta arbete delvis finansierats med bidrag från Singapore undervisningsministeriet (Tier 2: Grant ARC9 / 15), National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016) och NTU-NHG Aging Research Grant ( Grant ARG / 14017). Vi vill uttrycka vår tacksamhet och mest uppriktiga tack till Dr. Andrew Alpert och PolyLC team för vänligt försett oss med förpackningsmaterial som möjliggjort denna studie.

1. Förpackning Long-längd Anjon-utbyte (LAX) kapillärkolonn (Anmärkning: Även om den LAX kolonnen kan vara i hemmet förpackade som vi beskriver i detta protokoll, LAX kolonner är också kommersiellt tillgängliga, se tabell av Material och reagens för ytterligare detaljer). <ol><li> Suspendera 50 mg av svag anjonbytare packningsmaterial i 3,5 ml packningsbuffert (tabell 1) för att framställa uppslamningen. </li><li> Montera änden av kap...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+deamidation:+differentiation+of+glutamic+acid+and+gamma-glutamic+acid+in+peptides+by+electron+capture+dissociation.">Glutamine deamidation: differentiation of glutamic acid and gamma-glutamic acid in peptides by electron capture dissociation.</a> Anal Chem. 82 (9), 3606-3615 (2010).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal Biochem. 150 (1), 76-85 (1985).</li><li>Serra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+proteome+coverage+is+increased+by+unique+peptide+recovery+from+sodium+deoxycholate+precipitate.">Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate.</a> Anal Bioanal Chem. , 1-11 (2016).</li><li>Gallart-Palau, X., Serra, A., Sze, S. 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I denna video-artikeln presenterar vi en steg-för-steg-protokoll för LERLIC-MS / MS-3, till en metod att utföra en fördjupad karakterisering och för att noggrant bestämma graden av protein deamidering och de enzymatiska processer involverade i denna proteinmodifiering. LERLIC-MS / MS är baserad på användningen av en lång längd (50 cm) LAX enligt principen om elektrostatisk repulsion-hydrofil interaktions-kromatografi (ERLIC) 27. Användning av en kolonn med lån...

Deamidering är ett protein posttranslationell modifiering (PTM) som introducerar en negativ laddning till proteinet ryggraden genom modifiering av asparagin (Asn) och / eller glutamin (Gin) resterna 1. Denna modifikation samtidigt som de påverkar Asn-rester genererar isomera produkterna isoasparaginsyra (isoAsp) och n -asparaginsyra (Asp) vid en gemensam 3: 1-förhållande 2. Utan hinder, kan detta förhållande ändras genom ingripandet av reparation enzymet L-isoaspartyl metyltransferas (PIMT) 3, 4. På liknande sätt, deamidering av Gin-rester genererar den isomera gamma-glutaminsyra (γ-Glu) och alfa-glutaminsyra isoformer (α-Glu) vid en förväntad 1: 7-förhållande 3, 5, men detta förhållande kan förskjutas genom verkan av den allestädes närvarande enzym transglutaminas 2 och andra transglutaminaser, inklusive transglutaminas 1, ett enzyme nyligen identifierats som associerade med extracellulära vesiklar i hjärnan 6. Ursprunget för deamidering kan vara antingen spontan eller enzymatisk, den förra är särskilt vanligt på Gin-rester i vilka transglutaminaser och andra enzymer medierar inter / intra-molekylär tvärbindning via transamidering (se 3 för ytterligare detaljer om Gin transamidering och dess konsekvenser i flera kroniska och dödliga sjukdomar hos människan). Därför är deamidering en PTM som har en avgörande återverkan på strukturen och funktionen av påverkade molekyler 4, 7, 8 och kräver en fördjupad kemisk karakterisering 3 mot bakgrund av dess olika biokemiska konsekvenser inklusive dess tjänst som molekylärt klocka av åldrande 9 . Även deamidering av Asn rester har varit<html> relativt väl kännetecknas av bottom-up-hagelgevär proteomik 1, 10, deamidering av Gin-rester ändå inte har en lämplig karakterisering metod bortom den utmanande analys av modellföreningar genom elektronbaserad radikal fragmentering 11. Vi har nyligen utvecklat en ny en-dimensionen shotgun proteomics strategi (LERLIC-MS / MS) 3 som möjliggör separation av Gin och Asn deamidering isoformer från komplexa biologiska prover och modellföreningar i en enda analys. LERLIC-MS / MS är baserad på separationen av tryptiska digere peptider med användning av en lång längd (50 cm) jonbyteskolonn (LAX) arbetar på elektrostatisk repulsion-hydrofil interaktions-kromatografi (ERLIC) läge och kopplad till tandemmasspektrometri (LC- MS / MS). Denna nya analytiska strategi har använts för att karaktärisera och relativt kvantifiera omfattningen av varje deamiderade rest i humana hjärnvävnaderf &quot;&gt; 3. Trots det kommer det protokoll som beskrivs här ger video avbildning av LERLIC-MS / MS syftar att studera egenheter protein deamidering i den biokemiska sammanhang av intresse. ETIK ANALYS Alla förfaranden i detta protokoll har godkänts av Institutional Review Board i Nanyang Technological University i Singapore och har utförts i enlighet med de institutionella riktlinjerna.

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		</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

Karaktärisering av protein deamidering är absolut nödvändigt att dechiffrera roll (er) och möjligheter av detta protein posttranslationell modifiering (PTM) i human patologi och andra biokemiska sammanhang. För att utföra karaktärisering av protein deamidering, har vi nyligen utvecklat en ny lång längd elektrostatisk repulsion-hydrofil interaktionskromatografi-tandemmasspektrometri (LERLIC-MS / MS) metod som kan separera den glutamin (Gin) och asparagin (Asn) isoform produkterna av deamidering från modellföreningar för att mycket komplexa biologiska prover. LERLIC-MS / MS är därför den första hagelgevär proteomik strategi för separation och kvantifiering av Gin deamidering isoformer. Vi visar också, som en nyhet, att provet bearbetning protokoll som beskrivs här stabiliserar succinimid mellanliggande tillåter dess karakterisering av LERLIC-MS / MS. Tillämpning av LERLIC-MS / MS som visas i denna video artikeln kan bidra till att belysa den aktuella unknown molekylära kedjor av protein deamidering. Dessutom ger LERLIC-MS / MS ytterligare förståelse av de enzymatiska reaktioner som omfattar deamidering i distinkta biologiska bakgrunder.

Deamidering av Gin och Asn-rester anses vara en degenerativ proteinmodifiering (DPM) inblandad i flera kroniska och dödliga sjukdomar 14. Det har visat sig att denna PTM kan förutsäga halveringstid och nedbrytande tillstånd av antikroppar och andra molekyler i människokroppen och liknande biologiska bakgrunder 1, 15. Betydelsen av protein deamidering, i själva verket går utöver den biomedicinska sa...

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

Här presenterar vi en steg-för-steg-protokoll av den lång längd elektrostatisk repulsion-hydrofil interaktionskromatografi-tandemmasspektrometri metoden (LERLIC-MS / MS). Detta är en ny metod som gör det möjligt för första gången kvantifiering och karakterisering av glutamin och asparagin deamidering isoformer genom shotgun proteomics.

LERLIC-MS / MS för Djupgående Karakterisering och kvantifiering av glutamin och asparagin Deamidering i Shotgun proteomik

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

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Biochemistry

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Djupa proteomet profilering av isobariska märkning, omfattande vätskekromatografi, masspektrometri och programvara-assisted kvantifiering

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

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Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

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

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Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Quantification of Coenzyme A in Cells and Tissues

Kvantifiering av coenzym A i celler och vävnader

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

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TMT Provberedning för Proteomics Facility Submission och efterföljande dataanalys

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

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JUMPn: En strömlinjeformad applikation för protein-samuttryckskluster och nätverksanalys inom proteomik

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Hagelgevär Proteomics provbehandling automatiserad av en labbrobot med öppen källkod

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, ...

Shotgun Lipidomics of Rodent Tissues

Hagelgevär lipidomik av gnagare vävnader

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...