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

Questo lavoro è stato in parte sostenuto da sovvenzioni dal Ministero della Pubblica Istruzione di Singapore (Tier 2: Grant ARC9 / 15), Consiglio Nazionale delle Ricerche di Medicina di Singapore (NMRC-OF-IRG-0003-2016), e NTU-NHG Aging Research Grant ( concessione ARG / 14017). Vorremmo esprimere la nostra gratitudine e il più sincero ringraziamento al Dr. Andrew Alpert e la squadra PolyLC per ci ha gentilmente fornito il materiale di imballaggio che hanno reso possibile questo studio.

1. Imballo Long-length di scambio anionico (LAX) colonna capillare (Nota: Anche se la colonna LAX può essere in casa imballato come descriviamo in questo protocollo, colonne LAX sono anche disponibili in commercio, vedi Tabella dei Materiali e reagenti per ulteriori dettagli). <ol><li> Sospendere 50 mg di materiale da imballaggio scambio anionico debole in 3,5 mL di tampone imballaggio (Tabella 1) per preparare l&#39;impasto liquido. </li><li> Mo...

<ol>
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In questo video-articolo presentiamo un protocollo passo-passo di LERLIC-MS / MS 3, un metodo per eseguire la caratterizzazione approfondita e di determinare con precisione l&#39;entità del deammidazione proteine ei processi enzimatici coinvolti in questa modifica proteina. LERLIC-MS / MS è basato sull&#39;uso di un lungo lunghezza (50 cm) LAX secondo il principio della elettrostatico cromatografia interazione repulsione-idrofila (Erlic) 27. L&#39;utilizzo di una colonna...

Deamidation è una modificazione post-traduzionale di proteine (PTM) che introduce una carica negativa al backbone proteina mediante una modifica di asparagina (Asn) e / o glutammina (Gln) residui 1. Questa modifica mentre colpisce residui Asn genera prodotti isomerici acido isoaspartic (isoAsp) e n acido -aspartic (Asp) ad un comune rapporto 3: 1 2. Nonostante, questo rapporto può essere alterato dall&#39;intervento della riparazione enzima L-isoaspartyl metiltransferasi (PIMT) 3, 4. Analogamente, deamidation di residui Gln genera acido isomerica gamma-glutammico (γ-Glu) e isoforme di acido alfa-glutammico (α-Glu) ad un atteso rapporto 1: 3, 5 7, ma questo rapporto può essere spostata dall&#39;azione di l&#39;onnipresente enzima transglutaminasi 2 e altri transglutaminasi, compreso transglutaminasi 1, una enzyme recentemente identificato come associati con vescicole extracellulari nel cervello 6. L&#39;origine di deamidation può essere spontanea o enzimatica, il primo è particolarmente comune sui residui Gln in cui transglutaminasi e altri enzimi mediano reticolante / intra-molecolare tra via transamidazione (vedere 3 per ulteriori dettagli sulla Gln transammidazione e sue implicazioni in diversi cronica e malattie umane fatali). Pertanto, deamidation è un PTM che si ripercuote determinante sulla struttura e funzione delle molecole interessate 4, 7, 8 e richiede una caratterizzazione chimico approfondita 3 alla luce delle sue diverse conseguenze biochimiche compreso il servizio di clock molecolare dell&#39;invecchiamento 9 . Anche se deamidation dei residui Asn è stata<html> relativamente ben caratterizzato dal basso verso l&#39;alto fucile proteomica 1, 10, deammidazione di residui Gln ancora non ha un metodo di caratterizzazione adatto oltre l&#39;analisi impegnativo composti modello da elettron-based frammentazione radicale 11. Abbiamo recentemente sviluppato un nuovo unidimensionale proteomica fucile strategia (LERLIC-MS / MS) 3 che consente la separazione di Gln e Asn isoforme deamidazione da campioni biologici complessi e composti modello in una singola analisi. LERLIC-MS / MS è basato sulla separazione dei triptica digerito peptidi utilizzando una lunga lunghezza (50 cm) colonna di scambio ionico (LAX) operando in modalità elettrostatica repulsione-idrofila cromatografia a interazione (Erlic) ed accoppiata alla spettrometria di massa tandem (LC MS / MS). Questa nuova strategia analitico è stato usato per caratterizzare e quantificare relativamente l&#39;estensione di ciascuna residuo deamidato nei tessuti cerebrali umanif &quot;&gt; 3. Tuttavia, il protocollo descritto qui fornirà immagini video di LERLIC-MS / MS mirava a studiare le peculiarità di deamidation proteine nel contesto biochimica di interesse. ETICA DICHIARAZIONE Tutte le procedure di questo protocollo sono stati approvati dal comitato di revisione istituzionale della Nanyang Technological University di Singapore e sono state eseguite in conformità alle linee guida istituzionali.

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		<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

Caratterizzazione di deamidation proteina è indispensabile per decifrare il ruolo (s) e le potenzialità di questa proteina post-traslazionale modifica (PTM) nella patologia umana e in altri contesti biochimici. Al fine di eseguire la caratterizzazione di deamidation proteine, abbiamo recentemente sviluppato un nuovo lungo lunghezza elettrostatico interazione cromatografia-spettrometria di massa tandem repulsione-idrofilo (LERLIC-MS / MS) Metodo che può separare la glutammina (Gln) e asparagina isoforma (Asn) prodotti di deamidation da composti modello a campioni biologici molto complessi. LERLIC-MS / MS è, dunque, la prima strategia proteomica fucile per la separazione e la quantificazione di Gln isoforme deammidazione. Abbiamo anche dimostrato, come una novità, che il protocollo di trasformazione campione qui delineato stabilizza l&#39;intermedio succinimmide permettendo la sua caratterizzazione LERLIC-MS / MS. L&#39;applicazione di LERLIC-MS / MS come mostrato in questo articolo il video può aiutare a chiarire il momento sconosciutoarray n molecolari deamidation proteine. Inoltre, LERLIC-MS / MS fornisce ulteriore comprensione delle reazioni enzimatiche che comprendono deamidation in sfondi biologiche distinte.

Deamidation di residui Gln e Asn è considerata una modifica proteina degenerativa (DPM) implicati in numerose malattie croniche e mortali 14. È stato dimostrato che questo PTM può prevedere l&#39;emivita e degradative stati di anticorpi e altre molecole nel corpo umano e sfondi biologiche simili 1, 15. Il significato di deamidation proteine, infatti, va oltre il contesto biomedico, quindi questa modific...

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

Qui vi presentiamo un protocollo passo-passo del lungo lunghezza elettrostatico repulsione-idrofilo interazione cromatografia-spettrometria di massa tandem metodo (LERLIC-MS / MS). Questa è una nuova metodologia che consente per la prima volta quantificazione e caratterizzazione dei glutammina e asparagina isoforme deamidazione da proteomica fucile.

LERLIC-MS / MS per approfondita caratterizzazione e quantificazione di glutammina e asparagina deamidazione in Fucile 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

Proteoma profonda profilatura di etichettatura isobarico, vasto cromatografia liquida, spettrometria di massa e quantificazione assistita da Software

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

Ricerca

Biochimica

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

Caratterizzazione delle glicoproteine con la piega di immunoglobulina mediante cristallografia a raggi x e tecniche biofisiche

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

Tecnologia di Microarray di proteina extracellulare per il rilevamento di Throughput elevato di interazioni recettore-ligando bassa affinità

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

Rilevamento di siti di Ubiquitinazione proteica mediante arricchimento dei peptidi e spettrometria di massa

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

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

Entità DI legame SUMO (SUBE) come strumenti per l'arricchimento, l'isolamento, l'identificazione e la caratterizzazione del proteoma SUMO nel cancro al fegato

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

Quantificazione del Coenzevia A nelle cellule e nei tessuti

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

Preparazione del campione TMT per l'invio di strutture proteomiche e la successiva analisi dei dati

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: un'applicazione semplificata per il clustering di co-espressione proteica e l'analisi di rete in proteomica

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

Elaborazione dei campioni di Shotgun Proteomics automatizzata da un robot da laboratorio open source

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

Shotgun Lipidomics of Rodent Tissues

Lipidomica shotgun di tessuti di roditori

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