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

NOTA: Questo metodo è stato descritto in precedenza 12. 1. Proteina Isolamento Preparare 1 mg di proteina cellulare da parte delle cellule di lisi, preferibilmente con metodi fisici come stampa francese, perlina percosse, o ultrasuoni. Evitare di lisozima-lisi cellulare mediata perché l&#39;enzima si confondono misure di spettrometria di massa. 2. TCA precipitazione delle proteine Aggiungere acido tricloroacetico 1 volume (TCA) a 4 volumi di proteine ​​e freddo in ghiaccio per 10 minuti per precipitare le proteine. Centrifugare a 12.000 xg per 5 minuti a 4 ° C e rimuovere il surnatante. Risospendere il pellet in 1 ml di acetone ghiacciato e centrifugare a 12.000 xg per 5 minuti a 4 ° C. Rimuovere il surnatante e invertire il tubo sul banco per asciugare il pellet per 15 min. Pellets proteine ​​Conservare a -80 ° C. 3. Denaturare le proteine ​​e ridurre disolfuro Obbligazioni Resospendere proteine ​​a ~ 2 mg / ml in 500 microlitri tampone di denaturazione e riduzione (o 4 M urea o 3% SDS in 50 mM HEPES pH 8,5, 5 mM DTT). Facoltativamente, includere un inibitore della proteasi nel buffer. Incubare proteine ​​per 30 min a 56 ° C, seguita da 10 minuti a temperatura ambiente. 4. Aklylate gratis solfidrile Gruppi di interrompere Irreversibilmente legame disolfuro Formazione Preparare fresco 0.3 M iodoacetamide in acqua. ATTENZIONE! Iodoacetamide è altamente tossico. Aggiungere 25 microlitri 0,3 M iodoacetamide (15 mm concentrazione finale) a 500 microlitri di proteine ​​e incubare per 20 minuti al buio a temperatura ambiente. Placare iodoacetamide con l&#39;aggiunta di 10 ml di 300 mM DTT (5 mM concentrazione finale DTT). Conservare le proteine ​​alchilati a -80 ° C. 5. Digestione delle proteine TCA precipitare le proteine ​​(come descritto al punto 2) e risospendere in 1 ml di HEPES 50 mM (pH 8.2), 1 M urea. Preparare una soluzione stock di Lysyl endoproteinase (Lys-C) in acqua ad una concentrazione di 2 mg / mL e aggiungere 5 ml della soluzione proteica. Incubare la miscela per 16 ore a temperatura ambiente. Garantire la concentrazione finale Lys-C è 10 ng / ml e il rapporto proteina-to-lysC (w / w) è 1/50 a 1/200. Risospendere 20 mcg sequenziamento grado tripsina in 40 ml di 50 mM di acido acetico, aggiungere 5 ml (10 mg tripsina) alla Lys-C digest, e incubare per 6 ore a 37 ° C. Utilizzare la stessa concentrazione proteasi e rapporti proteasi-to-proteina per Lys-C come usato per la tripsina. 6. Fase inversa Peptide Estrazione <ol><li> Acidificare peptidi aggiungendo acido trifluoroacetico (TFA) a una concentrazione finale di 0,5% (pH ≈ 2). Attaccare una colonna C18 a un collettore di aspirazione. Usare la massima portata possibile per tutti i passaggi tranne carico e eluizione dei peptidi. </li><li> Colonna bagnato con 6 ml di acetonitrile (ACN). Lavare colonna con 6 ml 80% ACN, 0,1% TFA, poi equilibrare con 6 ml 0,1% TFA. Non permettere che lacolonna di funzionare a secco tra i passaggi. </li><li> Arrestare pressione di vuoto e carica 500 pg di peptidi sulla colonna ad una velocità di flusso di circa 1 ml / min. Una volta peptidi hanno legato alla colonna, riavvio vuoto e lavare con 6 ml 0,1% TFA, poi con 3 ml di tampone di acido citrico (0,09 M di acido citrico, 0.23 M Na 2 HPO 4, pH 5.5). Nota: gli importi più elevati di peptide possono essere etichettati ma la colonna C18 capacità di legame dovrebbero essere almeno due volte più elevate rispetto alla quantità peptide per evitare la perdita del campione. </li></ol> 7. On-colonna Peptide Labeling da riduttiva dimethylation (Redi Labeling) Eseguire questo passaggio sotto una cappa chimica cianuro di idrogeno viene rilasciato in bassa concentrazione durante il processo di etichettatura. <ol><li> Preparare 12 ml di &quot;leggere&quot; e &quot;pesanti&quot; buffer Redi a metilare ammine peptide gratuiti. Tampone Redi Luce si compone di 0,8% di formaldeide e 0,12 M sodiocianoboroidruro trasportano idrogeno in thEIR distribuzioni isotopiche naturali in tampone acido citrico. Tampone REDI pesante consiste di 0,8% di formaldeide deuterato e 0,12 M sodiocianoboroidruro deuterato in tampone acido citrico. </li><li> Incubare colonna contenente peptidi aggiungendo 10 ml luce o tampone Redi pesante alle colonne contenenti peptidi a portata di 1 ml / min e ripetere garantire un&#39;etichettatura completa. Lavare colonna con 6 ml 0,1% TFA e poi con 1 ml di 0,5% acido acetico. </li><li> Arrestare vuoto ed eluire prima peptidi marcati con 1 ml di 40% ACN, 0,5% di acido acetico, poi con 1 ml di 80% ACN, 0,5% di acido acetico utilizzando una portata di circa 0,5 ml / min. Se lo si desidera, misurare l&#39;efficienza etichettatura dei singoli campioni mediante spettrometria di massa prima della miscelazione campioni pesanti e leggeri (vedi &quot;Rappresentante dei risultati&quot;). Mescolare 01:01 campioni peptide pesanti e leggeri etichettati per essere quantificata mediante spettrometria di massa. </li></ol> 8. Peptide separato miscela in base pH fase reversibile (BPRP) Cromatografia &lt;/ P&gt; PH basico fase inversa (BPRP) cromatografia per separare la miscela di peptidi in più frazioni, che vengono analizzati in modo indipendente da LC-MS/MS per aumentare la copertura proteoma. <ol><li> Frazionare la miscela peptidica su una colonna C18-HPLC applicando un gradiente di concentrazione crescente ACN in 10 mM di bicarbonato di ammonio (pH 8). Iniziare con 5% (v / v) ACN per 5 min, aumentare al 35% ACN, in 60 minuti, e poi al 90% ACN in 1 min. Conservare il ACN 90% per 4 minuti prima di ridurre l&#39;ACN al 5% di riequilibrare la colonna per 9 min. Raccogliere 96 frazioni di pari volume in una piastra a 96 pozzetti (da A1 a H12). Monitorare il frazionamento utilizzando un rivelatore UV a 220 nm, mentre i peptidi sono eluizione largo della colonna (10-70 min per le condizioni qui descritte). </li><li> Combina frazioni da pozzi A1, C1, E1 e G1 (frazione A1), da pozzi B1, D1, F1, e H1 (frazione B1), da pozzi A2, C2, E2, e G2 (frazione A2) e di conseguenza per l&#39; restanti frazioni. Rimuovere il solvent utilizzando una centrifuga a vuoto. Peptidi Risospendere da frazioni A1, B2, A3, B4, A5, B6, A7, B8, A9, B10, A11 e B12 in 130 ml di 1 M urea/0.5% TFA e purificare usando StageTips come descritto al punto 9. Negozio frazioni B1, A2, B3, A4, B5, A6, B7, A8, B9, A10, B11 e A12 a -20 ° C. </li></ol> 9. Purify peptidi da Stop and Go estrazione (StageTips) Preparare C18-StageTip 7 microcolonne da imballaggio 200 ml punte per pipette con due C18 dischi con un diametro interno (ID) di 1,07 mm. Mettere Fase Consigli in tubi Eppendorf. Utilizzare una microcentrifuga per lavare punte con 130 ml di metanolo, quindi 130 ul 80% ACN, 0,5% di acido acetico. Equilibrare StageTips con 130 microlitri 0,1% TFA. Trasferire la miscela di peptidi a StageTips e lavare con 130 microlitri 0,1% TFA, quindi 40 microlitri 0,1% TFA, quindi 40 microlitri 0,5% di acido acetico. Eluire peptidi prima con il 20 microlitri 40% ACN, 0,5% di acido acetico, poi 20 l 80% ACN, 0,5% di acido acetico. Combina eluati unnd secco mediante filtrazione sotto vuoto. 10. Microcapillare LC-MS/MS <ol><li> Sciogliere peptidi in 1-5 ml di acido formico al 5%, 5% ACN ad una concentrazione di circa 1 mg / mL. Risolvere ~ 1 mg peptidi su un 100 pm × 20 cm C18-invertiti colonna HPLC in fase con gradiente 6-22% in ACN 0,125% acido formico ripartiti lungo 75 o 100 minuti ad una portata di ~ 300 Nl / min. </li><li> Identificare i peptidi utilizzando un LTQ Orbitrap Velos 12 o simile piattaforma liquido cromatografia-spettrometria di massa con uno spettrometro di massa che preveda ad alta risoluzione ed elevata accuratezza di massa. Azionare lo spettrometro di massa in modalità dati-dipendente con un full MS scansione (risoluzione di 60.000), acquisita nel analizzatore Orbitrap. Generare trappola ionica lineare MS / MS spettri per i 20 ioni più abbondanti rilevati nello spettro completo di MS. Impostare il controllo automatico del guadagno (AGC) target a 1 x 10 6 per la piena SM e 2000 per MS / MS. Impostare tempi massimi di accumulo di ioni a 1.000 msecper la SM e 150 msec per MS / MS. Escludi frammentate ioni peptide precursore di un&#39;ulteriore selezione da MS / MS per 20-60 sec. </li></ol> 11. MS / MS Acquisizione Dati Identificare peptidi confrontando file RAW spettri MS / MS ad un database teorico con un algoritmo come SEQUEST 8 utilizzando questi parametri (Tabella 1). Tabella 1. Peptide ricerca database dei parametri. <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> Parametri generali </td><td> digestione completamente trittico con un massimo di 2 perdere fratture </td></tr><tr height="19"><td> 25 ppm tolleranza ione precursore </td></tr><tr height="19"><td> 1.0 Da tolleranza agli ioni frammento </td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> Modifiche statici </strong&gt; </td><td> 57,02146 Da sulla cisteina, carboxyamidomethylation </td></tr><tr height="19"><td> 28,03130 Da sulla lisina e il peptide N-terminale, etichetta dimethylation luce </td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> Modifiche dinamici </td><td> Da 15,99,491 mila su metionina, ossidazione </td></tr><tr height="19"><td> 6,03766 Da sulla lisina e il peptide N-terminale, etichetta dimethylation pesante </td></tr></tbody></table><ol><li> Filtra peptidi ad aliquote scoperta falso 1% con un metodo quale il 9 strategia target-richiamo utilizzando un database di open reading frames negli orientamenti attuali e invertiti. </li></ol> 12. Peptide Quantificazione Calcolare le aree di coppie pesanti e leggeri di MS1 estratti ioni cromatogrammi (aree di picco MS1) e peptide segnale-rumore (S / N) Formati 10. Includi coppie peptide solo quando il loro segnale medio-to-noirapporto se è superiore a cinque. Quantificare abbondanza relativa di un peptide nei due campioni come il rapporto delle aree dei picchi MS1 di versioni pesanti e leggere dello stesso peptide (MS1 rapporto dell&#39;area di picco). Calcola abbondanze proteici relativamente come mediana rapporto area del picco MS1 per tutti i peptidi della proteina.

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Gli autori dichiarano di non interessi finanziari concorrenti o altri conflitti di interesse.

Numerosi punti fanno etichettatura isotopo stabile di peptidi utilizzando un metodo interessante per la proteomica quantitativa dimethylation riduttiva (etichettatura Redi): reagenti poco costosi di etichettatura (reagenti costano meno di $ 1 per esempio), velocità di reazione rapida (~ 10 min), assenza di prodotti collaterali, alte riproducibilità (figure 3, 4), prodotti di reazione stabili, capacità di utilizzare qualsiasi proteasi e ad alta efficienza di ionizzazione dei peptidi marcati. Etichetta...

Misurare differenze di concentrazione di molte proteine ​​tra i campioni complessi è una sfida centrale in proteomica. Sempre più spesso, questo viene fatto etichettando le proteine ​​in ogni campione con diversi tag isotopiche, mescolando i campioni, e l&#39;utilizzo di spettrometria di massa per quantificare le differenze di concentrazione. Esistono diversi metodi per l&#39;etichettatura isotopica stabile di proteine ​​e peptidi. 15 N etichettatura 1 e 2 SILAC introdurre etichette isotopiche metabolicamente in vivo, che iCAT 3, iTRAQ 4, e la riduzione dimethylation 5 aggiungere tag isotopi stabili dopo l&#39;estrazione delle proteine ​​e la digestione. Tra questi metodi, dimethylation riduttiva (etichettatura Redi) sta guadagnando popolarità come un poco costoso, metodo riproducibile per quantificare le differenze di concentrazione di proteine ​​in quasi ogni tipo di campione. Etichettatura REDI comporta peptidi che reagiscono con formaldeide per formare una base di Schiff, che viene poi ridottoda cianoboroidruro. Questa reazione dimethylates gruppi amminici liberi su N-Termini e laterali lisina catene e monomethylates proline N-terminale. Il protocollo qui descritto metila peptidi nel campione 1 con un&#39;etichetta &quot;luce&quot; utilizzando reagenti con atomi di idrogeno nella loro distribuzione isotopica naturale e campione 2 con un&#39;etichetta &quot;pesante&quot; utilizzando formaldeide deuterato e cianoboroidruro (Figura 1). Ogni gruppo amminico dimetilato su un peptide provoca una differenza di massa di 6,0377 Da tra luce e forme pesanti, che viene impiegata per distinguere tra i due moduli utilizzando uno spettrometro di massa. In particolare, abbondanze peptide relativi vengono quantificati come rapporto delle aree cromatogramma ionico MS1 estratti (MS1 rapporto dell&#39;area di picco) di luce e versione pesante per ogni coppia peptide ione. L&#39;abbondanza relativa di una proteina è calcolato come rapporto mediano area del picco MS1 tra tutti i peptidi della proteina. In questo rapporto, descriviamo un protocollo robusto per comportamentozione esperimenti di marcatura Redi da LC-MS/MS che include peptide estrazione a fase inversa in fase solida, etichettatura on-column Redi, peptide frazionamento da pH basico fase (BPRP) cromatografia inversa, e la purificazione di miscele di peptidi utilizzando StageTips (Figura 2) . Discutiamo vantaggi ei limiti di utilizzo etichettatura redi per proteomica quantitativa.

<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

Etichettatura isotopi stabili di peptidi di dimethylation riduttiva (etichettatura Redi) è un metodo per quantificare con precisione le differenze di espressione proteica tra campioni utilizzando la spettrometria di massa. Etichettatura Redi viene eseguita utilizzando sia regolare (luce) o deuterati (pesanti) forme di formaldeide e sodio cianoboroidruro di aggiungere due gruppi metilici ad ogni ammina libera. Qui mostriamo un protocollo robusto per l&#39;etichettatura Redi e comparazione quantitativa di miscele di proteine ​​complesse. Campioni di proteine ​​di confronto vengono digeriti in peptidi, etichettati a portare luce o tag metilici pesanti, mescolato, e co-analizzati mediante LC-MS/MS. Abbondanze relative di proteina sono quantificate confrontando le aree di picco di ioni cromatogramma delle versioni etichettate pesanti e leggeri del peptide costituente estratto dalla piena MS spettri. Il metodo qui descritto comprende la preparazione del campione mediante estrazione in fase solida in fase inversa, etichettatura Redi on-column di peptidi, peptide frazionamento da pH rev basefase ersed (BPRP) cromatografia, e StageTip depurazione peptide. Discutiamo vantaggi ei limiti di etichettatura Redi rispetto ad altri metodi per isotopi stabili incorporazione. Evidenziamo nuove applicazioni utilizzando etichettatura Redi come un metodo rapido, economico e preciso per confrontare le abbondanze di proteine ​​in quasi ogni tipo di campione.

Abbiamo valutato l&#39;accuratezza, la precisione e riproducibilità di etichettatura Redi utilizzando Saccharomyces cerevisiae e Clostridium phytofermentans lisati cellulari interi. In primo luogo abbiamo quantificato l&#39;efficienza etichettatura Redi di un mix di C. phytofermentans lisati proteici da cellulosa (pesante etichetta, H) e glucosio (etichetta luce, L) culture. Quando filtrato per un peptide tasso di scoperta di falsi 1%, questo campione conteneva 11.194 sequenze peptidiche unic...

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

Etichettatura isotopi stabili di peptidi da dimethylation riduttiva (etichettatura Redi) è un rapido strategia economica per proteomica accurate di massa a base di spettrometria-quantitativi. Qui mostriamo un metodo efficace per la preparazione e l&#39;analisi di miscele proteiche che utilizzano l&#39;approccio Redi che può essere applicato a quasi qualsiasi tipo di campione.

La proteomica quantitativa, utilizzando riduttiva dimethylation per Stable Isotope Labeling

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

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Research

JoVE Journal

Chemistry

16.0K Views.  Genoscope.  Etichettatura isotopi stabili di peptidi da dimethylation riduttiva (etichettatura Redi) è un rapido strategia economica per proteomica accurate di massa a base di spettrometria-quantitativi. Qui mostriamo un metodo efficace per la preparazione e l'analisi di miscele proteiche che utilizzano l'approccio Redi che può essere applicato a quasi qualsiasi tipo di campione. 

Video: 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.

Su larga scala e non mirato Profiling Metabolomica di siero da Ultra Performance Liquid Chromatography-Spettrometria di Massa (UPLC-MS)

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

Ricerca

Chimica

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.

Miglioramento in gel riduttive solfo-glycomics β-eliminazione completa per O-linked e di spettrometria di massa

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.

Esame quantitativa e qualitativa delle interazioni particella-particella colloidale Uso Probe Nanoscopia

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.

Un quantitativo Glycomics e proteomica combinata strategia Purificazione

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.

Norme per quantitativa Metalloproteomic analisi utilizzando 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 ...

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.

Peptidi e proteine quantificazione utilizzando 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 ...

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.

Proteomica di Top-down su larga scala usando la spettrometria totale in Tandem elettroforesi capillare zona

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.

Multiplexing di campioni automatizzato utilizzando l'etichettatura isotopica precursore combinata e il tagging isobarico (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.

Sospensioni acquose stabili di cluster di ferrite di manganese con dimensioni e composizione su scala nanometrica sintonizzabili

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

Identificazione degli organi primari nelle piante medicinali mediante marcatura con 13C6-glucosio

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.

Autore in primo piano: Profilazione degli organi primari delle piante utilizzando la marcatura del 13C6-glucosio e 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.

13Marcatura del C6-glucosio associata a LC-MS: identificazione degli organi primari delle piante nella sintesi di metaboliti secondari

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