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 arbeidet ble delvis støttet med tilskudd fra Singapore Ministry of Education (Tier 2: Grant ARC9 / 15), National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016), og NTU-NHG Aging stipend ( Grant ARG / 14017). Vi ønsker å uttrykke vår takknemlighet og mest oppriktige takk til Dr. Andrew Alpert og PolyLC team for vennlig gitt oss med den originale emballasjen gjort mulig denne studien.

1. Emballasjen Long-lengde Anion-utveksling (LAX) kapillarkolonnen (Merk: Selv om LAX kolonnen kan være i hjemmet pakket som vi beskriver i denne protokollen, LAX kolonner er også kommersielt tilgjengelig, se tabell over Materialer og reagenser for ytterligere detaljer). <ol><li> Suspender 50 mg svak anionbytter pakkemateriale i 3,5 ml pakkingsbuffer (tabell 1) for å fremstille oppslemmingen. </li><li> Monter enden av den kapillære kolonne (50...

<ol>
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I denne video-artikkelen presenterer vi en trinn-for-trinn-protokollen for LERLIC-MS / MS 3, en fremgangsmåte utføre grundig karakterisering og for nøyaktig å bestemme graden av protein deamidering og de enzymatiske prosesser som er involvert i denne proteinmodifisering. LERLIC-MS / MS er basert på bruken av en lang lengde (50 cm) LAX henhold til prinsippet om elektrostatiske avstøtning-hydrofile interaksjoner kromatografi (ERLIC) 27. Bruken av en lang lengde kolonne...

Deamidering er et protein posttranslasjonell modifikasjon (PTM) som innfører en negativ ladning til selve proteinryggraden gjennom modifikasjon av asparagin (Asn) og / eller glutamin (Gin) residier 1. Denne modifikasjon mens påvirker Asn-restene genererer isomere produkter isoaspartic syre (isoAsp) og n-asparaginsyre (Asp) ved et felles forholdet 3: 1 2. Ikke desto mindre kan dette forhold endres ved intervensjon av reparasjon enzymet L-isoaspartyl metyltransferase (PIMT) 3, 4. På lignende måte, deamidering av Gln-rester genererer isomere gamma-glutaminsyre (γ-Glu) og alfa-glutaminsyre-isoformer (α-Glu) til en forventet 1: 7-forhold 3, 5, men dette forhold kan forskyves ved virkningen av den allestedsnærværende enzymet transglutaminase 2 og andre transglutaminases, inkludert transglutaminase en, en enzyme nylig identifisert som assosiert med ekstracellulære vesikler i hjernen 6. Opprinnelsen av deamidering kan enten være spontan eller enzymatisk, den førstnevnte er spesielt vanlig på Gln-rester i hvilke transglutaminases og andre enzymer som medierer den inter / intra-molekylære tverrbinding via transamidering (se 3 for nærmere informasjon om Gln transamidering og dens konsekvenser i flere kroniske og dødelige sykdommer hos mennesker). Derfor er deamideringen en PTM som har en avgjørende ettervirkning på strukturen og funksjonen til berørte molekyler 4, 7, 8 og krever en grundig kjemisk karakterisering 3 i lys av dens forskjellige biokjemiske konsekvenser med sin tjeneste som molekylære klokke av aldring 9 . Selv om deamidering av Asn-restene har vært<html> forholdsvis godt karakterisert ved nedenfra og opp hagle proteomforskning 1, 10, deamidering av Gln-rester fremdeles ikke har en passende karakteristikk metode utover utfordrende analyse av modellforbindelser ved elektron-radikal fragmentering 11. Vi har nylig utviklet en ny ett-dimensjon hagle proteomforskning strategi (LERLIC-MS / MS) 3 som muliggjør separasjon av Gin og Asn deamideringsseter isoformer fra komplekse biologiske prøver og modell-forbindelser i en enkelt analyse. LERLIC-MS / MS er basert på separasjon av tryptiske peptider nedbrytes ved hjelp av en lang lengde (50 cm) ionebytterkolonne (SFO) arbeider på modus elektrostatiske avstøtning-hydrofile interaksjoner kromatografi (ERLIC) og koplet til tandem massespektrometri (LC MS / MS). Denne nye analyse strategi har blitt brukt for å karakterisere og forholdsvis kvantifisere utstrekningen av hver isoaspartatholdig rest i menneskelige hjernen vevf &quot;&gt; 3. Ikke desto mindre vil protokollen beskrevet her gi videobilder av LERLIC-MS / MS forsøkte å studere de særegenheter protein deamidering i den biokjemiske forbindelse av interesse. ETIKK ERKLÆRING Alle prosedyrer i denne protokollen er godkjent av Institutional Review Board av Nanyang Technological University i Singapore, og er utført i henhold til de institusjonelle retningslinjer.

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			<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 av protein deamidering er viktig å dechiffrere rollen (e) og potensialene til dette proteinet posttranslasjonell modifikasjon (PTM) i humane patologi, og andre biokjemiske sammenhenger. For å utføre karakterisering av protein deamidering, har vi nylig utviklet en ny lang lengde elektrostatiske avstøtning-hydrofil interaksjonskromatografi-tandem massespektrometri (LERLIC-MS / MS) -metoden som kan skille glutamin (Gin) og asparagin (Asn) isoform produkter av deamidering fra modellforbindelser for å svært komplekse biologiske prøver. LERLIC-MS / MS er derfor den første hagle proteomforskning strategi for separasjon og kvantifisering av GLN deamideringsseter isoformer. Vi viser også, som en nyhet, at prøvebehandlingsprotokollen beskrevet her stabiliserer succinimidet mellomliggende tillater dens karakterisering av LERLIC-MS / MS. Bruk av LERLIC-MS / MS som vist i denne videoen artikkelen kan bidra til å belyse tiden unknown molekylære sammensetninger av protein deamidering. I tillegg gir LERLIC-MS / MS ytterligere forståelse av de enzymatiske reaksjoner som omfatter deamidering i forskjellige biologiske bakgrunn.

Deamidering av Gln og Asn-restene er ansett som en degenerativ proteinmodifisering (DPM) implisert i en rekke kroniske og dødelige sykdommer 14. Det er blitt vist at denne PTM kan forutsi halveringstid og nedbrytnings tilstander av antistoffer og andre molekyler i det menneskelige legeme og lignende biologiske bakgrunn 1, 15. Betydningen av protein deamidering, faktisk går utover biomedisinsk sammenheng,...

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 presenterer vi en trinn-for-trinn-protokollen for lang lengde elektrostatiske avstøtning-hydrofil interaksjonskromatografi-tandem massespektrometri (LERLIC-MS / MS) -metoden. Dette er en roman metode som gjør det mulig for første gang kvantifisering og karakterisering av glutamin og asparagin deamideringsseter isoformer av hagle proteomikk.

LERLIC-MS / MS for inngående karakterisering og kvantifisering av glutamin og asparagin Deamidering i hagle Proteomics

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

Research

JoVE Journal

Biochemistry

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

Dyp proteom profilering av isobar merking, omfattende flytende Ture, massespektrometri og Software-baserte kvantifisering

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

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Karakteristikk av glykoproteiner med immunglobulin Fold av Røntgenkrystallografi og Biofysiske teknikker

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Ekstracellulære Protein Microarray teknologi for høy gjennomstrømning påvisning av lav affinitet reseptor-Ligand interaksjoner

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

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Påvisning av protein ubiquitination nettsteder av peptid berikelse og masse spektrometri

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

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

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SUMO-bindende enheter (SUBEs) som verktøy for berikelse, isolasjon, identifisering og karakterisering av SUMO proteom i leverkreft

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

Kvantifisering av koenzym A i celler og vev

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

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

TMT Prøve forberedelse for proteomics facility innsending og påfølgende dataanalyse

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

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

JUMPN: En strømlinjeformet applikasjon for klynger med proteinkouttrykk og nettverksanalyse i proteomikk

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

Hagle proteomics prøvebehandling automatisert av en åpen kildekode lab robot

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

Shotgun Lipidomics of Rodent Tissues

Hagle lipidomikk av gnagervev

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