Name: bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome
Uploaded: 2014-03-07T14:30:00
Description: Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Forfatterne takker Dr. Kent Seeley, direktør for Center for Drug Discovery og Innovation (CDDI) Proteomics Core Facility ved University of South Florida for brug af denne facilitet. Dette arbejde blev støttet af NIH / NIDCD tilskud R01 DC004295 til BHAs

Etiske retningslinjer Eksperimenter med mus væv blev godkendt af University of South Florida Institutional Animal Care og brug Udvalg (Protokoller 3931R, 3482R) som angivet i henhold til retningslinjerne for National Institutes of Health. 1.. Protein Extraction <ol><li> Isoler cochlear sensorisk epithel fra 16 30 dage gamle (P30) CBA / J-mus og opbevares ved -80 ° C. </li><li> På dagen for forsøget vaskes vævet med 500 pi 1x phosphatpufret saltvand (PBS). Cen...

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<html> De vigtigste skridt til at maksimere protein identifikation fra den cochlear sensoriske epithel er: 1) brug af flere endoproteinaser for fordøjelsen, 2) brug af flere separeringsteknikker, og 3) udnyttelse af en høj opløsning massespektrometer. Anvendelsen af ​​flere enzymer øger antallet af peptider og forbedrer proteinsekvens dækning dermed forbedre antallet af identificerede proteiner fra cochlear væv. Trypsin, de mest almindeligt anvendte protease giver en effektiv og specifik spaltning af proteiner,...

Proteomics er studiet af komplekse biologiske systemer ved at analysere protein udtryk, funktion, modifikationer og interaktioner 1. Der er blevet anvendt flere metoder til proteomanalyse af det indre øre, herunder antistof microarray 2 todimensional gelelektroforese 3-5, og DIGE 6. Der er dog kun identificeret et begrænset antal proteiner og karakteriseret 2,7-10, i forhold til de over 10.000 gener og udtrykte sekvens tags (EST) identificeret i det indre øre 11,12, MS er den mest udbredte og omfattende teknik i proteomics til protein karakterisering. Analyse af komplekse proteom prøver, såsom cochlea, kan være udfordrende. Kombinationen af flere separationsteknikker MS muliggør identifikation af et større antal af peptider og proteiner på grund af en øget dynamisk koncentrationsområde og maksimal kapacitet 13. Multidimensional chromatograPHY reducerer meget komplekse proteinblandinger ved at tillade anvendelse af forskellige adsorption mekanismer. Der er to almindeligt anvendte MS proteomanalyse tilgange, shotgun og bottom-up proteomics. I shotgun proteomics, er en blanding af intakte proteiner fordøjes enzymatisk og separeret ved hjælp af multidimensionel kromatografi med stærk kationbytningskromatografi (SCX) efterfulgt af omvendt fase væskekromatografi (RPLC) 14,15. De separerede peptider er udsat for tandem MS og databasesøgning 15. En stor fordel ved denne teknik er, at tusindvis af proteiner kan identificeres i en enkelt analyse, og teknikken er bedre egnet til membranproteiner. I bottom-up approach proteinet blanding adskilles, sædvanligvis ved en-eller todimensional elektroforese, og de individuelle proteinbånd eller pletter udskåret og fordøjet med et enzym, såsom trypsin, hvilket sædvanligvis resulterer i multiple peptider. Men en anden nyerely udviklet elektroforese tilgang, som anvendes i bottom-up proteomics, er GELFrEE. Denne teknik fraktionerer protein prøver i flydende fase og gør dem mindre kompleks inden analyse. Denne teknik er reproducerbare, tilbyder højt proteinindhold opsving, og reducerer distribution af høj rigelige proteiner i komplekse protein prøver 16. Peptider, som følge af separerede proteiner, analyseres ved MS, ved at bruge peptidmassefingeraftryk eller tandem MS (MS / MS), for at skabe sekvenstags for databasesøgning 17-19. Nogle af de store fordele ved at bruge bottom-up tilgang er evnen til at opnå høj opløsning separationer og omfattende protein dækning. Bottom-up proteomics er den mest udbredte teknik proteomics 20 dermed findes flere bioinformatik værktøjer. Desuden kan proteiner separeres i en kompleks blanding før fordøjelse, så der er en større chance for identifikation. En af de store udfordringeri at bruge det indre øre for proteom analyse er dens lille størrelse, begrænset tilgængelighed og celletype mangfoldighed 21. Derudover har vigtige proteiner, der kendetegner dets funktionalitet, såsom ionkanaler, transportører og receptorer er membranproteiner, der kan være vanskelige at isolere 22. Således filter-aided prøveforberedelse (FASP) er en fordel for proteom analyser af væv, der er begrænset for protein udvinding og som kræver rengøringsmidler til at opløse membraner 23. Denne filtrering giver mulighed for MS-analyse af membran og opløselige proteiner og for evnen til at isolere peptider fra lavmolekylære forureninger 23,24. Den nuværende protokol beskriver almindeligt anvendte proteom tilgange, som er kombineret og modificeret til at analysere både opløselige og membranproteiner og for at maksimere antallet af protein-id&#39;er fra cochlea sensoriske epithel. Vi vil beskrive bruge shotgun proteomics med FASP multi-fordøjeion, ionbytningskromatografi, højopløsnings-MS og dataanalyse. Desuden vil vi beskrive bottom-up proteomics med GELFrEE, FASP multi-fordøjelse, høj opløsning MS, og dataanalyse.

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			<td height="20" style="height:20px;width:264px;">8% Tris-acetate cartridge&#160;</td>
			<td style="width:159px;">Protein Discovery</td>
			<td style="width:133px;">42103</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Honeywell</td>
			<td>015-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AEBSF</td>
			<td>Calbiochem</td>
			<td>101500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>AC16861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aprotinin</td>
			<td>Calbiochem</td>
			<td>616370</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ASB-14&#160;</td>
			<td>Calbiochem</td>
			<td>182750-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>BioRad</td>
			<td>500-0112</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">C18 column&#160;</td>
			<td>New Objective</td>
			<td>A25112</td>
			<td>75 &#956;m &#215; 10 cm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DC Protein Assay</td>
			<td>BioRad</td>
			<td>500-0116</td>
			<td>Microplate Assay Protocol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endoproteinase Lys-C</td>
			<td>Sigma-Aldrich</td>
			<td>P3428</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FASP Protein Digestion Kit</td>
			<td>Protein Discovery</td>
			<td>44250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid&#160;</td>
			<td>Fluka</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GELFrEE Fractionation System</td>
			<td>Protein Discovery</td>
			<td>42001</td>
			<td>GELFrEE 8100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leupeptin</td>
			<td>Calbiochem</td>
			<td>108975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MacroSpin Column</td>
			<td>The Nest Group</td>
			<td>SMM SS18V</td>
			<td>Silica C18</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcystin</td>
			<td>Calbiochem</td>
			<td>475815</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pepstatin</td>
			<td>Sigma-Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysulfoethyl A Column</td>
			<td>The Nest Group</td>
			<td>202SE0503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonic Dismembrator&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-338-53</td>
			<td>Model 100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
			<td>Proteomics Grade</td>
		</tr>
	</tbody>
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bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome

Proteomics er en almindeligt anvendt fremgangsmåde, der kan give indsigt i komplekse biologiske systemer. Cochlear sensoriske epithel indeholder receptorer, der transducerer den mekaniske energi af lyd i en elektro-kemisk energi behandles af de perifere og centrale nervesystem. Adskillige proteom teknikker er blevet udviklet til at studere cochlear indre øre, såsom todimensional forskel gelelektroforese (2D-DIGE) antistof mikroarray, og massespektrometri (MS). MS er den mest omfattende og alsidige værktøj i proteomics og i forbindelse med separation metoder kan give en dybdegående proteom af biologiske prøver. Separation metoder kombineret med MS har evnen til at berige protein prøver, påvisning med lav molekylvægt og hydrofobe proteiner og identificere lave talrige proteiner ved at reducere proteomet dynamikområde. Forskellige fordøjelse strategier kan anvendes til hel lysat eller fraktioneret proteinlysat at øge peptid og proteinsekvens dækning. Udnyttelse af forskellige separationsteknikker, herunder stærk kationbytter (SCX), omvendt fase (RP), og gel-elueret væskefraktion fastklemning elektroforese (GELFrEE) kan anvendes til at reducere prøve kompleksitet forud for MS-analyse for protein identifikation.

For at opnå den mest omfattende proteom af cochlear sensoriske epithel er hurtig væv dissektion påkrævet forud for protein udvinding og prøveforberedelse. To proteom teknikker kan anvendes, shotgun og bottom-up proteomics. For at fremstille prøver til shotgun proteomics blev FASP fordøjelse, der anvendes som vist i figur 1. Den FASP metode giver mulighed for koncentration af proteiner, fjernelse af vaskemidler, og fordøjelsen af ​​proteiner ved hjælp af flere enzymer. Der var to dobbelt for...

Watch this Scientific Journal Video about Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome at JoVE.com

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Bottom-up og Shotgun Proteomics at identificere en omfattende Cochlear Proteome

Proteomanalyse af cochlear sensoriske epithel kan være en udfordring på grund af sin lille størrelse, og fordi membranproteiner er vanskeligt at isolere og identificere. Både membranen og opløselige proteiner, kan identificeres ved at kombinere flere præparative metoder og separationsteknikker sammen med høj opløsning massespektrometri.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

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Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Ortogonale Proteinoprensning Faciliteret af en lille Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi screening for at identificere Postembryonic fænotyper i C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolering af ribo Bundet Gryende polypeptider<em&gt; In vitro</em&gt; Til at identificere Translationelle Pause steder langs mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Produktion af<em&gt; Xenopus tropicalis</em&gt; Egg uddragene Identificer mikrotubuli-associerede RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Capture Compound massespektrometri - Et kraftfuldt værktøj til at identificere nye C-di-GMP effektorproteiner

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Ikke-enzymatisk, Serumfrit Tissue Culture of Pre-invasive brystlæsioner til spontane generation af Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Xenopus laevis as a Model to Identify Translation Impairment

Xenopus laevis as a Model to Identify Translation Impairment

Xenopus laevis som en model til at identificere Oversættelse Nedskrivning

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...