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

HINWEIS: Diese Methode wurde bereits beschrieben 12. 1. Proteinisolierung Herstellung von 1 mg Zellprotein durch Lysieren von Zellen, vorzugsweise durch physikalische Methoden wie Französisch Presse Wulst Schlagen oder Ultraschallbehandlung. Vermeiden Lysozym-vermittelte Zell-Lyse, weil das Enzym Massenspektrometrie Messungen zu verwechseln. 2. TCA-Fällung von Proteinen 1 Volumen Trichloressigsäure (TCA) bis 4 Volumen Protein-und Chill auf Eis für 10 min, um Proteine ​​zu fällen. Zentrifuge bei 12.000 × g für 5 min bei 4 ° C und Entfernen des Überstandes. Resuspendieren des Pellets in 1 ml eiskaltem Aceton und Zentrifuge bei 12.000 × g für 5 min bei 4 ° C. Entfernen Sie den Überstand und das Röhrchen auf der Bank, um das Pellet für 15 Minuten trocknen. Speicher Proteinpellets bei -80 ° C. 3. Proteine ​​denaturieren und die Reduzierung von Disulfidbrücken ReProteine ​​aussetzen, um ~ 2 mg / ml in 500 ul Denaturierung und Reduktion Puffer (entweder 4 M Harnstoff, 3% SDS in 50 mM HEPES pH 8,5, 5 mM DTT). Optional umfassen einen Proteaseinhibitor in Puffer. Proteine, Inkubation 30 min bei 56 ° C, gefolgt von 10 min bei Raumtemperatur. 4. Aklylate freien Sulfhydrylgruppen irreversibel stören Disulfidbindungsbildung Bereiten Sie frischen 0,3 M iodoacetamide in Wasser. ACHTUNG! Iodacetamid ist hochgiftig. Hinzufügen von 25 &amp;mgr; l 0,3 M Iodacetamid (15 mM Endkonzentration) zu 500 ul Protein und Inkubation für 20 min im Dunkeln bei Raumtemperatur. Quench iodoacetamide durch Zugabe von 10 ul 300 mM DTT (5 mM DTT endgültige Konzentration). Speichern alkylierten Proteinen bei -80 ° C. 5. Protein Verdauung TCA-Ausfällung Proteine ​​(wie in Schritt 2 beschrieben) und Resuspendieren in 1 ml 50 mM HEPES (pH 8,2), 1 M Harnstoff. Bereiten Sie eine Stammlösung von Lysyl endoproteinase (Lys-C) in Wasser bei einer Konzentration von 2 ug / ul und mit 5 &amp;mgr; l der Proteinlösung. Inkubieren der Mischung für 16 Stunden bei Raumtemperatur. Sicherzustellen, dass die endgültige Lys-C-Konzentration 10 ng / &amp;mgr; l, und das Protein-zu-LysC-Verhältnis (w / w) 1/50 bis 1/200 ist. Resuspendieren 20 ug Trypsin sequencing grade in 40 ul 50 mM Essigsäure werden 5 ul (10 ug Trypsin) an die Lys-C-Verdau und Inkubation für 6 h bei 37 ° C. Verwenden die gleiche Proteasekonzentration und Protease-zu-Protein-Verhältnisse für die Lys-C wie Trypsin verwendet. 6. Umkehrphasen-Peptid-Extraktion <ol><li> Anzusäuern Peptide durch Zugabe von Trifluoressigsäure (TFA) bis zu einer Endkonzentration von 0,5% (pH ≈ 2). Bringen Sie eine C18-Säule einer Extraktion vielfältig. Verwenden Sie die höchste Durchflussrate für alle Schritte außer Be-und Elution der Peptide. </li><li> Nass Säule mit 6 ml Acetonitril (ACN). Waschen Säule mit 6 ml 80% ACN, 0,1% TFA, dann ins Gleichgewicht mit 6 ml 0,1% TFA. Lassen Sie dasSpalte trocken Schritte laufen. </li><li> Stoppen Vakuumdruck und Last 500 ug von Peptiden auf die Säule bei einer Fließgeschwindigkeit von etwa 1 ml / min. Nachdem Peptide auf die Säule gebunden Neustart Vakuum und wäscht mit 6 ml 0,1% TFA, dann mit 3 ml Zitronensäurepuffer (0,09 M Zitronensäure, 0,23 M Na 2 HPO 4, pH 5,5). Hinweis: Höhere Peptid-Mengen können markiert werden, aber die Bindungskapazität C18-Säule sollte mindestens zwei-fach höher als die Menge an Peptidprobenverlust zu vermeiden. </li></ol> 7. On-Spalte der Peptidmarkierung durch reduktive Dimethylierung (Redi Labeling) Führen Sie diesen Schritt unter einer chemischen Abzugshaube als Cyanwasserstoff in geringer Konzentration während des Markierungsprozesses freigesetzt. <ol><li> Bereiten Sie 12 ml &quot;Licht&quot; und &quot;schwer&quot; Redi Puffer Peptid freien Amine methylieren. Licht Redi Puffer besteht aus 0,8% Formaldehyd und 0,12 M Natriumcyanoborhydrid Durchführung Wasserstoffatome in their natürliche Isotopenverteilungen in Zitronensäure-Puffer. Schwere Redi Puffer besteht aus 0,8% deuteriertes Formaldehyd und 0,12 M deuteriertem Natriumcyanoborhydrid in Zitronensäure-Puffer. </li><li> Inkubieren Spalte Peptide durch Zugabe von 10 ml entweder leicht oder schwer Redi Puffer zu den Peptid-enthaltende Spalten am Flussrate von 1 ml / min, und wiederholen Sie die vollständige Kennzeichnung zu gewährleisten. Waschkolonne mit 6 ml 0,1% TFA und dann mit 1 ml 0,5% Essigsäure. </li><li> Stoppen Vakuum und markierte Peptide eluieren zuerst mit 1 ml 40% ACN, 0,5% Essigsäure, dann mit 1 ml 80% ACN, 0,5% Essigsäure mit einer Fließgeschwindigkeit von etwa 0,5 ml / min. Wenn gewünscht, messen Sie die Markierungseffizienz der einzelnen Proben mittels Massenspektrometrie vor dem Mischen der schweren und leichten Proben (siehe &quot;Repräsentative Ergebnisse&quot;). Mix 1.01 schweren und leichten markiertes Peptid Proben massenspektrometrisch quantifiziert werden. </li></ol> 8. Separate Peptidmischung von Basic pH Reversed Phase (BPRP) Chromatographie &lt;/ P&gt; Basischen pH-Reversed-Phase (BPRP) gereinigt, um die Peptid-Gemisch in mehrere Fraktionen, die unabhängig von LC-MS/MS analysiert werden Proteom-Abdeckung zu erhöhen trennen. <ol><li> Fraktionierung der Peptidmischung auf einer C18-HPLC-Säule durch Anlegen eines Gradienten mit zunehmender ACN-Konzentration in 10 mM Ammoniumbicarbonat (pH 8). Beginnen mit 5% (v / v) ACN für 5 min, zu erhöhen, um 35% ACN in 60 min, und dann auf 90% ACN in 1 min. Bewahren Sie die 90% ACN für 4 min vor Verringerung der ACN zu 5% wieder ins Gleichgewicht die Spalte für 9 min. Sammeln 96 Fraktionen von gleichem Volumen in einer 96-Well-Platte (A1 bis H12). Überwachung der Fraktionierung unter Verwendung eines UV-Detektors bei 220 nm, während Peptide Elution von der Säule (10-70 min für den hier beschriebenen Bedingungen). </li><li> Kombinieren Fraktionen aus Vertiefungen A1, C1, E1 und G1 (Fraktion A1) aus den Vertiefungen B1, D1, F1 und H1 (Fraktion B1) aus den Vertiefungen A2, C2, E2 und G2 (Fraktion A2) und demgemäß für die restlichen Fraktionen. Entfernen Sie die Zahlungsfähigkeitt mit einer Vakuumzentrifuge. Resuspendieren Peptide von Fraktionen A1, B2, A3, B4, A5, B6, A7, B8, A9, B10, A11 und B12 in 130 ul 1 M urea/0.5% TFA und Reinigung mit StageTips wie in Schritt 9 beschrieben. Shop Fraktionen B1, A2, B3, A4, B5, A6, B7, A8, B9, A10, B11, A12 und bei -20 º C. </li></ol> 9. Purify Peptiden durch Stop and Go Extraction (StageTips) Bereiten C18-StageTip 7 Mikrosäulen durch die Verpackung 200 ul Pipettenspitzen mit zwei C18-Festplatten mit einem Innendurchmesser (ID) von 1,07 mm. Setzen Bühnen Tipps in Eppendorf-Röhrchen. Verwenden Sie einen Mikro um Tipps mit 130 ul Methanol, dann 130 ul 80% ACN, 0,5% Essigsäure waschen. Ins Gleichgewicht StageTips mit 130 ul 0,1% TFA. Peptidgemisch zu übertragen StageTips und wäscht mit 130 ul 0,1% TFA, dann wurden 40 ul 0,1% TFA, dann 40 &amp;mgr; l 0,5% Essigsäure. Peptide eluieren zuerst mit 20 ul 40% ACN, 0,5% Essigsäure, dann 20 ul 80% ACN, 0,5% Essigsäure. Kombinieren Sie ein Eluatend trockenen durch Vakuumfiltration. 10. Mikrokapillar LC-MS/MS <ol><li> Lösen Peptide in 1-5 ul 5% Ameisensäure, 5% ACN in einer Konzentration von etwa 1 ug / ul. Beheben ~ 1 &amp;mgr; g Peptide auf eine 100 &amp;mgr; m × 20 cm C18-Umkehrphasen-HPLC-Säule mit einem Gradienten von 6-22% ACN in 0,125% Ameisensäure über 75 oder 100 min aufgebracht mit einer Flussrate von ca. 300 Nl / min. </li><li> Identifizieren Peptide mit Hilfe eines LTQ Orbitrap Velos 12 oder ähnliche Flüssigchromatographie-Massenspektrometrie-Plattform mit einem Massenspektrometer bietet hohe Auflösung und hohe Massengenauigkeit. Betreiben Sie das Massenspektrometer in den datenabhängigen Modus mit einem vollen MS-Scan (Auflösung von 60.000) in der Orbitrap Analysator erworben. Generieren linearen Ionenfalle MS / MS-Spektren für die 20 in der Voll MS-Spektrum erkannt häufigsten Ionen. Einstellen der automatischen Verstärkungsregelung (AGC) Ziele zu 1 × 10 6 für die volle MS und 2.000 für MS / MS. Stellen Sie maximale Ionenakkumulationszeiten bis 1000 msfür MS und 150 ms für MS / MS. Ausschließen fragmentierten Peptid-Vorläufer-Ionen von der weiteren Auswahl von MS / MS für 20-60 sek. </li></ol> 11. MS / MS-Datenerfassung Peptide zu identifizieren durch Vergleichen MS / MS-Spektren Rohdaten zu einem theoretischen Datenbank mit einem Algorithmus, wie SEQUEST 8 unter Verwendung dieser Parameter (Tabelle 1). Tabelle 1. Peptid Datenbanksuchparameter. <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> Allgemeine Parameter </td><td> vollständig tryptischen Verdau mit bis zu 2 verpasst Spaltungen </td></tr><tr height="19"><td> 25 ppm Vorläuferionen Toleranz </td></tr><tr height="19"><td> 1.0 Da Fragmentionentoleranz </td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> Statische Änderungen </sTrong&gt; </td><td> 57,02146 Da auf Cystein, Carboxyamidomethylierung </td></tr><tr height="19"><td> 28,03130 Da auf Lysin und dem Peptid N-Terminus, Licht Dimethylierung Label </td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> Dynamische Änderungen </td><td> 15,99491 Da an Methionin, Oxidation </td></tr><tr height="19"><td> Da auf 6,03766 Lysin und dem Peptid N-Terminus, schwere Dimethylierung Label </td></tr></tbody></table><ol><li> Filter Peptide zu einer falschen Erkennungsrate von 1% mit einem Verfahren wie dem Ziel-Decoy-9-Strategie unter Verwendung einer Datenbank von offenen Leserahmen in den tatsächlichen und Orientierungen umgekehrt. </li></ol> 12. Peptid-Quantifizierung Berechnen Sie die Bereiche der schweren und leichten Paare von MS1 extrahiert Ionenchromatogramme (MS1 Peakflächen) und Peptid-Signal-zu-Rauschen (S / N)-Verhältnisse 10. Fügen Peptidpaare nur, wenn ihre durchschnittlichen Signal-noise Verhältnis über fünf. Quantifizierung der relativen Menge eines Peptids in den zwei Proben als das Verhältnis der Peakflächen MS1 ​​von schweren und leichten Varianten des gleichen Peptids (MS1 Peakflächenverhältnis). Berechnen relativer Häufigkeiten Protein als Median MS1 ​​Peakflächenverhältnis für alle Peptide in dem Protein.

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Die Autoren erklären, keine konkurrierenden finanziellen Interessen oder andere Interessenkonflikte.

Einige Punkte machen stabilen Isotopenmarkierung von Peptiden mittels reduktiven Dimethylierung (Redi Kennzeichnung) eine attraktive Methode für die quantitative Proteomik: kostengünstige Markierungsreagenzien (Reagenzien kostet weniger als $ 1 pro Probe), schnelle Reaktionsgeschwindigkeit (~ 10 min), das Fehlen von Nebenprodukten, hohe Reproduzierbarkeit (Fig. 3, 4), stabiler Reaktionsprodukte, die Fähigkeit, eine beliebige Protease verwendet, und hohe Ionisierungseffizienz von markierten Peptiden. ...

Messkonzentrationsunterschiede vieler Proteine ​​zwischen komplexen Proben ist eine zentrale Herausforderung in der Proteomik. Zunehmend wird dies durch Markierung von Proteinen in jeder Probe mit unterschiedlichen Isotopen-Tags, die Kombination der Proben und mit Hilfe der Massenspektrometrie, um Konzentrationsunterschiede zu quantifizieren getan. Es gibt verschiedene Verfahren für stabile Isotopenmarkierung von Proteinen und Peptiden. Kennzeichnung 15 N 1 und 2 SILAC vorstellen Isotopenmarkierungen metabolisch in vivo, während iCAT 3, iTRAQ 4, 5 und Reduktion Dimethylierung nach der Proteinextraktion und Verdauung hinzufügen stabilen Isotopen-Tags. Unter diesen Verfahren wird die reduktive Dimethylierung (Redi-Markierung) an Popularität gewinnt als eine preiswerte, reproduzierbare Methode zur Proteinkonzentration Unterschiede in fast jeder Art von Probe zu quantifizieren. REDi Markierung beinhaltet die Umsetzung von Peptiden mit Formaldehyd zu einer Schiffschen Base, die dann reduziert wird, zu bilden,von Cyanoborhydrid. Diese Reaktion dimethylates freien Aminogruppen auf N-Termini und Lysin-Seitenketten und monomethylates N-terminale Prolin. Das hier beschriebene Protokoll methyliert Peptide in Probe 1 mit einem &quot;light&quot;-Label unter Verwendung von Reagenzien mit Wasserstoffatomen in ihrer natürlichen Isotopenverteilung und Probe 2 mit einer &quot;schweren&quot; Etikett mit deuteriertem Formaldehyd und Cyanoborhydrid (Abbildung 1). Jeder dimethylierten Aminogruppe auf einem Peptid zu einer Massendifferenz von 6,0377 Da zwischen leichten und schweren Formen, die verwendet wird, um zwischen den beiden Formen mit einem Massenspektrometer zu unterscheiden. Insbesondere werden relativen Häufigkeiten Peptid als das Verhältnis der MS1 extrahiert Ionenchromatogramm Bereiche (Peakflächenverhältnis MS1) von leichten und schweren Version für jedes Peptid Ionenpaar quantifiziert. Die relative Menge eines Proteins wird als Median MS1 ​​Peakflächenverhältnis aller Peptide in dem Protein errechnet. In diesem Bericht beschreiben wir ein robustes Protokoll für das Verhaltening Redi Markierungsexperimente mit LC-MS/MS, die Umkehrphasen-Peptid-Festphasenextraktion, On-Column-Kennzeichnung redi-, Peptid-Fraktionierung durch basischen pH-Reversed-Phase (BPRP) Chromatographie und Reinigung der Peptidgemische mit StageTips (Abbildung 2) umfasst . Wir diskutieren Vor-und Nachteile der Verwendung von Redi Kennzeichnung für die quantitative Proteomik.

<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

Stabile Isotopenmarkierung von Peptiden durch reduktive Dimethylierung (Redi Kennzeichnung) ist eine Methode, um genau zu quantifizieren Proteinexpressionsunterschiede zwischen den Proben mit Hilfe der Massenspektrometrie. Redi Kennzeichnung erfolgt mit entweder regelmäßig (Licht) oder deuteriertes (Schwer-) Formen von Formaldehyd und Natriumcyanoborhydrid zu zwei Methylgruppen an jedem freien Amin hinzufügen. Hier zeigen wir, einen robusten Protokoll für REDi Kennzeichnung und quantitativen Vergleich von komplexen Proteingemischen. Proteinproben für den Vergleich sind in Peptide, beschriftet, entweder leicht oder schwer Methyl-Tags, gemischt und mittels LC-MS/MS analysiert Co-tragen verdaut. Relative Protein Häufigkeiten werden durch den Vergleich der Ionenchromatogramm Peakflächen von schweren und leichten markierten Versionen der Peptidbestandteil von der vollen MS-Spektren extrahiert quantifiziert. Das hier beschriebene Verfahren umfasst die Probenvorbereitung durch Umkehrphasen-Festphasenextraktion, On-Column-Redi Markierung von Peptiden, Peptid-Fraktionierung durch basischen pH-reversed-Phase (BPRP) Chromatographie und StageTip Peptidreinigung. Wir diskutieren Vorteile und Grenzen der redi Kennzeichnung gegenüber anderen Methoden zur stabilen Isotopen Gründung. Wir zeigen neue Anwendungen mit Redi Kennzeichnung als eine schnelle, kostengünstige und genaue Methode, um die Proteinhäufigkeiten in fast jeder Art von Probe zu vergleichen.

Wir untersuchten die Genauigkeit, Präzision und Reproduzierbarkeit der redi Kennzeichnung mit Saccharomyces cerevisiae und Clostridium phytofermentans Ganzzelllysaten. Wir haben zuerst quantifiziert die redi Markierungseffizienz aus einer Mischung von C phytofermentans Proteinlysate aus Cellulose (schwere beschriftet, H) und Glucose (Licht-Label, L) Kulturen. Wenn ein Peptid False Discovery Rate 1% gefiltert, enthielt diese Probe 11.194 einzigartige Peptidsequenzen mit einer Redi Markierungse...

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

Stabile Isotopenmarkierung von Peptiden durch reduktive Dimethylierung (Redi-Markierung) ist eine schnelle, kostengünstige Strategie für genaue Massenspektrometrie-basierte quantitative Proteomik. Hier zeigen wir ein robustes Verfahren für die Aufbereitung und Analyse von Proteingemischen mit Hilfe der redi Ansatz, der auf fast jedem Probentyp angewandt werden kann.

Quantitative Proteomics Mit reduktive Dimethylierung Stabile Isotope Labeling

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

quantitative-proteomics-using-reductive-dimethylation-for-stable

Research

JoVE Journal

Chemistry

16.0K Aufrufe.  Genoscope.  Stabile Isotopenmarkierung von Peptiden durch reduktive Dimethylierung (Redi-Markierung) ist eine schnelle, kostengünstige Strategie für genaue Massenspektrometrie-basierte quantitative Proteomik. Hier zeigen wir ein robustes Verfahren für die Aufbereitung und Analyse von Proteingemischen mit Hilfe der redi Ansatz, der auf fast jedem Probentyp angewandt werden kann. 

Serie: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Large Scale Non gezielte Metabolomic Profiling von Serum von Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Forschung

Chemie

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Verbesserte In-Gel-reduktive β-Elimination für umfassende O-gebundene und Sulfo-Glykomik durch Massenspektrometrie

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Quantitative und qualitative Untersuchung der Partikel-Partikel Wechselwirkungen mit Kolloidales Probe Nanoskopie

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

Eine quantitative Glycomics und Proteomics Kombinierte Reinigungsstrategie

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

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Standards für die quantitative Metalloproteomic Analyse mit Größenausschluss-ICP-MS

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

Peptide and Protein Quantification Using Automated 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 result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptid und Protein Quantifizierung mit Hilfe automatisierter 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 ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Groß angelegte Top-Down-Proteomik mit Kapillare Zone Elektrophorese Tandem-Massenspektrometrie

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

Automatisiertes Sample-Multiplexing mit kombinierter Vorläufer-Isotop-Etikettierung und isobaischer Kennzeichnung (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Stabile wässrige Suspensionen von Manganferritclustern mit abstimmbarer nanoskaliger Dimension und Zusammensetzung

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

Identifizierung von primären Organen in Heilpflanzen mittels 13C 6-Glukose-Markierung

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Autor im Rampenlicht: Profilerstellung von primären Organen von Pflanzen mittels 13C 6-Glukose-Markierung und LC-MS

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13C 6-Glukosemarkierung im Zusammenhang mit LC-MS: Identifizierung pflanzlicher Primärorgane bei der Synthese von Sekundärmetaboliten

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...