Name: quantitative proteomics using reductive dimethylation for stable isotope labeling
Uploaded: 2014-07-01T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

BEMÆRK: Denne metode er tidligere beskrevet 12. 1. Protein Isolation Klargør 1 mg celleprotein ved lyserende celler, fortrinsvis ved fysiske fremgangsmåder, såsom franske presse bead beating eller sonikering. Undgå lysozym-medieret cellelysis fordi enzymet vil forvirre massespektrometri målinger. 2.. TCA-præcipitation af proteiner Tilsæt 1 volumen trichloreddikesyre (TCA) til 4 volumener pro...

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Flere punkter gør stabil isotop mærkning af peptider ved hjælp af reduktiv dimethylation (redi mærkning) en attraktiv metode til kvantitative proteomics: billige mærkningsreagenser (reagenser koste mindre end $ 1 pr prøve), hurtig reaktionshastighed (~ 10 min), fravær af sideprodukter, høje reproducerbarhed (figur 3, 4), stabile reaktionsprodukter, evne til at bruge enhver protease og høj ionisering effektivitet af mærkede peptider. Kemisk mærkning af Redi er også fordelagtig i forhold til m...

Måling koncentrationsforskelle af mange proteiner mellem komplekse prøver er en central udfordring i proteomics. Stigende grad, dette bliver gjort af mærkning af proteiner i hver prøve med forskellige isotopiske tags, der kombinerer prøverne, og ved hjælp af massespektrometri til at kvantificere koncentrationsforskelle. Der findes flere metoder til stabil isotop-mærkning af proteiner og peptider. 15 N mærkning 1 og SILAC 2 indfører isotopiske etiketter metabolisk in vivo, mens ICAT 3, iTRAQ 4 og reduktion dimethylation 5 tilføje stabile isotop tags efter proteinekstraktion og fordøjelse. Blandt disse metoder er reduktiv dimethylation (redi mærkning) stigende popularitet som en billig og reproducerbar metode til at kvantificere proteinkoncentration forskelle i næsten enhver form for prøve. Redi mærkning involverer reagerende peptider med formaldehyd til dannelse af en Schiff-base, som derefter reduceresved cyanoborhydrid. Denne reaktion dimethylates frie aminogrupper på N-Termini og lysinsidekæder og monomethylates N-terminale proliner. Protokollen beskrevet her methylerer peptider i prøve 1 med en &quot;let&quot; label anvendelse af reagenser med hydrogenatomer i deres naturlige isotopiske distribution og prøve 2 med en &quot;tung&quot; etiket ved hjælp deutereret formaldehyd og natriumcyanborhydrid (figur 1). Hver dimethylerede aminogruppe på et peptid resulterer i en massiv forskel på 6,0377 Da mellem lette og tunge former, der anvendes til at skelne mellem de to former ved hjælp af et massespektrometer. Specifikt relative peptid hyppighed kvantificeret som forholdet MS1 ekstraherede ionkromatogram områder (MS1 toparealforhold) af lette og tunge version for hvert peptid ionpar. Den relative forekomst af et protein er beregnet som den mediane MS1 toparealforhold blandt alle peptider i proteinet. I denne rapport beskriver vi en robust protokol for adfærdning Redi mærkning eksperimenter ved LC-MS/MS, der omfatter omvendt-fase peptid fast fase-ekstraktion, Redi mærkning af kolonnen, peptid fraktionering ved basisk pH-omvendt fase (BPRP) kromatografi og oprensning af peptidblandinger hjælp StageTips (figur 2) . Vi diskuterer fordele og begrænsninger ved brug af Redi mærkning for kvantitative proteomics.

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

Stabil isotop mærkning af peptider ved reduktiv dimethylation (redi mærkning) er en metode til præcist at kvantificere protein udtryk forskelle mellem prøver ved anvendelse af massespektrometri. Redi mærkningen udføres ved hjælp af enten regelmæssige (lys) eller deutereret (tunge) former af formaldehyd og natriumcyanoborhydrid at tilføje to methylgrupper til hver fri amin. Her demonstrerer vi en robust protokol for redi mærkning og kvantitativ sammenligning af komplekse proteinblandinger. Protein prøver til sammenligning er fordøjet i peptider, mærket til at bære enten lette eller tunge methyl tags, blandet, og co-analyseret ved LC-MS/MS. Relative protein mængderne kvantificeres ved at sammenligne ionkromatogram Toparealerne for tunge og lette mærkede versioner af det konstituerende peptid ekstraheres fra den fulde MS-spektre. Den her beskrevne metode omfatter prøveforberedelse ved omvendt fase fastfaseekstraktion, Redi mærkning på kolonne af peptider, peptid fraktionering ved basisk pH reversed-fase (BPRP) kromatografi og StageTip peptidoprensning. Vi diskuterer fordele og begrænsninger ved Redi mærkning med hensyn til andre metoder for stabil isotop inkorporering. Vi fremhæver nye applikationer ved hjælp redi mærkning som en hurtig, billig og præcis metode til at sammenligne protein overflod i næsten enhver form for prøve.

Vi evaluerede nøjagtighed, præcision og reproducerbarhed Redi mærkning hjælp Saccharomyces cerevisiae og Clostridium phytofermentans helcellelysater. Vi først kvantificeret Redi mærkning effektiviteten af en blanding af C. phytofermentans proteinlysater fra cellulose (tung mærket H) og glucose (lys etiket, L) kulturer. Når filtreret til et peptid falsk opdagelse 1%, denne prøve indeholdt 11.194 unikke peptidsekvenser med en 98% redi mærkning effektivitet. Ufraktioneret S. cerevis...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Kvantitative Proteomics anvendelse af reduktiv Dimethylation for mærkning med stabile isotoper

Stabil isotop mærkning af peptider ved reduktiv dimethylation (redi mærkning) er en hurtig, billig strategi for nøjagtige massespektrometri-baserede kvantitative proteomics. Her demonstrerer vi en robust metode til forberedelse og analyse af proteinblandinger hjælp Redi tilgang, der kan anvendes til næsten enhver prøvetype.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

CN-GELFrEE - Klar Native Gel-elueres væskefraktion Fastklemning Elektroforese

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

En Kvantitativ Glycomics og Proteomics Kombineret Purification strategi

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Standarder for Kvantitative Metalloproteomic Analyse Brug Size Udelukkelse ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Meget Stabilt, Functional Behårede Nanopartikler og Biopolymerer fra træfibre: Mod bæredygtig Nanoteknologi

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Peptid og Protein kvantificering ved hjælp af automatiseret Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Hurtig Nanoprobe Signal forstærkning af In Situ guld nanopartikel syntese

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...