Wir danken den finanziellen Unterstützung von Genom Kanada und Genom British Columbia für Operationen (204PRO) und Technologieentwicklung (214PRO) durch das Genom Innovationen Netz (GIN). Wir danken der Drug Discovery-Plattform am Research Institute von der McGill University Health Center für die Nutzung der MALDI-TOF-Instrument für die Dreharbeiten. H.L ist dankbar für die Unterstützung von ein Postdoc-Stipendium von der National Science and Engineering Research Council of Canada (NSERC). C.H.B ist dankbar für die Unterstützung von Leading Edge Endowment Fund (LEEF). C.H.B. ist dankbar für Unterstützung von den Segal McGill Lehrstuhl für Molekulare Onkologie an der McGill University (Montreal, Quebec, Kanada). M.X.C. und C.H.B. sind dankbar für Unterstützung von Warren Y. Soper Charitable Trust und Alvin Segal Family Foundation die Jewish General Hospital (Montreal, Quebec, Kanada).

die Beträge der unten beschriebenen Reagenzien basieren auf der Messung von 20 Patienten Plasmaproben. Das Protokoll Nachstehend folgt die Richtlinien von der University of Victoria &#39; s Humanforschung Ethikkommission.
 1. Generation von Ang ich im menschlichen Plasma <ol><li>tauen Plasmaproben (&#8805; 200 µL) in einem Raum Temperatur Wasser Bad für 5 min, und setzen Sie dann die Proben auf Eis bis vollständig aufgetaut.</li>
	<li>Transfer 200 µL jeder Plasma Probe manuell zu trennen Brunnen von einem 1,1 mL Deep-Well-Platte (Probenteller), und die Platte in einer Zentrifuge für 10 min bei 2 ° C und 1278 X g Zentrifugieren</li>
	<li>Verwenden eine automatisierte Liquid handling-System, um eine 500 Fmol/µL Ang I NAT standard-Lösung zur Vorbereitung sechs Kalibrator Lösungen mit Huhn Eiweiß Albumin seriell zu verdünnen (0,1, 0,2, 0,6, 1,9, 5.7, 17,2 Fmol/µL).</li>
	<li>Jedem Kalibrator, ein gut und 125 µL Generation Pipette 200 µL Puffer Probenteller. 
	Hinweis: Die CEWA in Phosphat-gepufferter Kochsalzlösung (PBS) Puffer muss am Tag des Experiments frisch zubereitet werden.</li>
	<li>Mit einer automatisierten Flüssigkeit handling-System, in einer neuen Platte mix 125 µL überstand Plasma oder 125 µL CEWA mit PBS-Puffer mit 25 µL Ang ich Generation Puffer, enthält 1 M Tris, 0,2 mM Ethylenediaminetetraacetic Säure (EDTA) und 1 mM Phenylmethylsulfonyl Fluorid (PMSF). 
	Hinweis: Bereiten Sie die Ang ich Generation Puffer durch das Mischen von 1 M Tris/0,2 mM EDTA wässrigen Puffers (passen Sie auf pH 5,5 mit Essigsäure) und eine PMSF Lösung (100 mM in Methanol). Beide Lösungen können bis zu 1 Monat bei 4 ° c-Mix der beiden Lösungen am Tag des Experiments gelagert werden.</li>
	<li>Übertragen die Lösungen automatisch in eine 96-Well-Platte, 3 Wiederholungen pro Lösung, 34 µL pro Bohrloch. Inkubieren Sie die Platte bei 37 ° C für 3 h</li>
</ol> 2. Immuno-Anreicherung von Ang ich mittels Antikörper-beschichtete Perlen <ol><li>Konjugation des Antikörpers auf magnetische Protein G Perlen Hinweis: Konjugation des Antikörpers mit den Perlen erfolgt manuell in den 3 h Ang ich Generation Zeit auf die Tag des Experiments. Für andere Analyte könnte die konjugierten Perlen in PBS-Puffer mit 0,015 % CHAPS (PBSC) bei 4 ° C für drei Monate oder länger, je nach Eigenschaften und Stabilität des Antikörpers gespeichert werden können.
	<ol><li>Transfer 110 µL Wulst Gülle (reicht für 20 Proben zu messen und damit eine standard 6-Punkt-Kurve) in einer 1,5 mL-Tube. Waschen Sie die Perlen siebenmal mit 1 mL 25 % Acetonitril/peripherer und dreimal mit 1 mL peripherer. Verwenden Sie ein Magnetstativ, um die Perlen zwischen jedem Waschschritt pellet. Entfernen Sie die Waschpuffer nach der letzten Wash 
		Hinweis: Waschen 7 Mal mit 25 % ist Acetonitril/peripherer entscheidend zur Beseitigung der MS-inkompatible Additive im Wulst Gülle, wie z. B. Tween-20. In den ausgewählten Perlen gibt es keine solche Zusätze, dieses umfangreiche Waschschritt möglicherweise nicht notwendig sein.</li>
		<li>Die Perlen in 110 µL peripherer aufzuwirbeln, und fügen Sie 110 µL Anti-Ang ich Antikörper (endgültige Antikörperkonzentration: 100 µg/mL). Mischen Sie die Perlen und die Lösung durch pipettieren und inkubieren Sie sie bei Raumtemperatur für 1 h, 8 u/min drehen.</li>
		<li>Waschen die Perlen 3 Mal mit 1 mL peripherer und in 1100 µL peripherer Aufschwemmen. 
		Hinweis: Wenn keine Wulst-Lösung in die Kappe des Rohres während der Inkubation geflossen ist, spin-down die Lösung unter Verwendung einer Benchtop-Zentrifuge bei 2680 X g.</li>
		<li>Übertragen die Wulst-Lösung manuell in eine 96-Well-Platte (Perle standard Platte). Verwenden Sie die automatisierte Flüssigkeit Handlingsystem, aliquoten die Perlen an der gleichen 96-Well-Platte, 120 µL pro Well.</li></ol></li> <li>Immuno-Anreicherung an den Perlen <ol><li>nach 3 h Ang Generation Zeit, lege ich die Inkubation Platte auf Eis für 10 min in die Generation der Ang I. beenden</li>
		<li>Automatisch Verdünnen der SIS Peptid-Stammlösung (10 Pmol/μL) 100fach mit PBS Puffer, und automatisch weitere Aliquot der stabilen Isoptope standard Peptid Verdünnung auf eine 96-Well-PCR-Platte. 1,5 µL einer stabilen Isotop Normen (SIS) Peptid-Lösung (mit 100 Fmol) in jede Vertiefung der Inkubation Platte übertragen und mischen Sie es mit der Plasma-Proben oder der CEWA in PBS-Puffer.</li>
		<li>Automatisch den Inhalt der Inkubation Platte auf die Wulst Lösung, 10 mL pro Bohrloch übertragen, mix.</li>
		<li>Die Platte bei 4 ° C für 1 h beim Drehen mit 8 u/min inkubieren.</li>
		<li>Die Perlen dreimal waschen automatisch mit 5 mM Ammonium Bicarbonat (AmBic) Lösung, 100 µL pro Bohrloch pro Waschgang. Nach der letzten Wäsche Aufschwemmen der Perlen in 7 µL 5 mM AmBic Lösung in jede Vertiefung. Verwenden Sie ein Magnet, um die Perlen nach dem letzten Wash nach unten ziehen</li>
	</ol></li></ol> 3. Übertragung von Perlen auf eine MALDI Zieltafel und Hinzufügen von Matrix-Lösung <ol><li>Transfer 7 µL des Schlickers Wulst automatisch auf eine MALDI-Zieltafel mit einer Spotgröße von 2.600 µm. Lassen Sie die Perlen austrocknen. 
	Hinweis: Ein kleiner USB-betriebenen Ventilator kann verwendet werden, um die Perle, die Trocknung beschleunigen.</li>
	<li>Automatisch hinzufügen 2 µL der α-Cyano-4-Hydroxycinnamic Säure (HCCA)-Matrixlösung (mit 3 mg/mL HCCA, 1,8 mg/mL Ammonium Citrat, 70 % Acetonitril und 0,1 % Trifluoroacetic Säure) aus der Matrix gut auf jede Probe vor Ort auf die Zieltafel .</li>
</ol> 4. Übernahme durch ein MALDI-TOF-MS und Datenanalyse-Signal <ol><li>Analyse der Probe Flecken mit einem MALDI-TOF-Instrument mit positiven Reflektor-Modus. Führen Sie interne Kalibrierung Daten Glättung und Grundlinie Subtraktion automatisch mit herstellerspezifischen Software. 
	Hinweis: Die Betriebsart (positiv/negativ, lineare/Reflektor) für MALDI-TOF-Messung hängt von der Ziel-Peptide oder Proteine.</li>
	<li>Die relative Response Ratio (Nat/SIS Intensitätsverhältnis) zu berechnen und vergleichen Sie es mit der Standardkurve bestimmen die ang ich Konzentration in jeder Probe. Berechnen Sie PRA nach Gleichung (1), wo Δt Ang ich Generation repräsentiert die Zeit für die Generation der Ang I. 
	PRA = [Ang ich] / Δt Ang ich Generation (1)</li></ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Affinity+Capture+and+On-Tip+Digestion+to+Accurately+Quantitate+in+Vivo+Deamidation+of+Therapeutic+Antibodies.">Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate in Vivo Deamidation of Therapeutic Antibodies.</a> Anal. Chem. , (2016).</li><li>Razavi, M., Leigh Anderson, N., Pope, M. E., Yip, R., Pearson, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+precision+quantification+of+human+plasma+proteins+using+the+automated+SISCAPA+Immuno-MS+workflow.">High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.</a> N. Biotechnol. 33 (5 Part A), 494-502 (2016).</li><li>Razavi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+SISCAPA+Quantitation+of+Peptides+from+Human+Plasma+Digests+by+Ultrafast,+Liquid+Chromatography-Free+Mass+Spectrometry.">High-Throughput SISCAPA Quantitation of Peptides from Human Plasma Digests by Ultrafast, Liquid Chromatography-Free Mass Spectrometry.</a> J. Proteome Res. 11 (12), 5642-5649 (2012).</li><li>Anderson, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SISCAPA+Peptide+Enrichment+on+Magnetic+Beads+Using+an+In-line+Bead+Trap+Device.">SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device.</a> Mol. &amp; Cell. Proteomics. 8 (5), 995-1005 (2009).</li><li>Parker, C. E., Pearson, T. W., Anderson, N. L., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometry-based+clinical+proteomics+-+a+review+and+prospective.">Mass-spectrometry-based clinical proteomics - a review and prospective.</a> The Analyst. 135 (8), 1830-1838 (2010).</li><li>Reid, J. D., Holmes, D. T., Mason, D. R., Shah, B., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+Development+of+an+Immuno+MALDI+(iMALDI)+Mass+Spectrometry+Assay+for+the+Diagnosis+of+Hypertension.">Towards the Development of an Immuno MALDI (iMALDI) Mass Spectrometry Assay for the Diagnosis of Hypertension.</a> J. Am. Soc. Mass Spectrom. 21 (10), 1680-1686 (2010).</li><li>Mason, D. R., Reid, J. D., Camenzind, A. G., Holmes, D. T., Borchers, C. 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Proteomics. 10 (1), 20 (2013).</li><li>Jiang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+immuno+tandem+mass+spectrometry+(iMALDI)+assay+for+EGFR+diagnosis.">Development of an immuno tandem mass spectrometry (iMALDI) assay for EGFR diagnosis.</a> Proteomics Clin Appl. 1, (2007).</li><li>Jiang, J., Parker, C. E., Fuller, J. R., Kawula, T. H., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Immunoaffinity+Tandem+Mass+Spectrometry+(iMALDI)+assay+for+Detection+of+Francisella+tularensis.">An Immunoaffinity Tandem Mass Spectrometry (iMALDI) assay for Detection of Francisella tularensis.</a> Analytica chimica acta. 605 (1), 70-79 (2007).</li><li>Popp, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+assay+for+the+clinical+measurement+of+plasma+renin+activity+by+immuno-MALDI+(iMALDI).">An automated assay for the clinical measurement of plasma renin activity by immuno-MALDI (iMALDI).</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1854 (6), 547-558 (2015).</li><li>Schoenherr, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+monoclonal+antibodies+for+SISCAPA+assays+using+a+magnetic+bead+processor+and+liquid+chromatography-selected+reaction+monitoring-mass+spectrometry.">Automated screening of monoclonal antibodies for SISCAPA assays using a magnetic bead processor and liquid chromatography-selected reaction monitoring-mass spectrometry.</a> J Immunol Methods. 353 (1-2), 49-61 (2010).</li><li>Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+quantitation+and+identification+of+peptides+and+proteins.">Methods of quantitation and identification of peptides and proteins.</a> US patent. , (2010).</li><li>Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+quantitation+and+identification+of+peptides+and+proteins.">Methods of quantitation and identification of peptides and proteins.</a> Canadian patent CA. , (2011).</li><li>Wei&#223;, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+and+measure-mass+spectrometry-based+immunoassays+in+biomarker+research.">Catch and measure-mass spectrometry-based immunoassays in biomarker research.</a> Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1844 (5), 927-932 (2014).</li><li>Nelson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+fragments:+Hope+and+hype.">Antibody fragments: Hope and hype.</a> mAbs. 2 (1), 77-83 (2010).</li><li>Gupta, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+an+aptamer+for+use+as+an+affinity+reagent+with+MS:+PCSK9+as+an+example+protein.">An evaluation of an aptamer for use as an affinity reagent with MS: PCSK9 as an example protein.</a> Bioanalysis. 8 (15), 1557-1564 (2016).</li></ol>

C.H.B hält das Patent auf die iMALDI-Technologie.

Im Vergleich zu herkömmlichen MS-basierte Protein Quantifizierung, nutzt iMALDI Antikörper zur Bereicherung der Analyten und reinigen sie von komplexen Proben, daher macht es möglich, Proteine oder Peptide in geringen Konzentrationen zu quantifizieren. Ein wichtiger Schritt in das iMALDI-Protokoll ist die Immuno-Anreicherung der Ziel-Peptide. Zu diesem Zweck sollten Antikörper mit hoher Spezifität und Affinität ausgewählt werden. In SISCAPA wurde berichtet, dass Antikörper Affinitäten bei 10-9 M oder ...

Massenspektrometrie ist ein unverzichtbares Werkzeug in quantitative Proteomik geworden. Massenspektrometrie kann die Massen der Zielproteine oder Peptide bestimmen, also die erhaltenen Analyten Signale können hochspezifische im Vergleich zu Immunoassays. Zwei sind Ionisation, Electrospray und MALDI, am häufigsten Methoden zur Erkennung von Proteinen und Peptiden1,2,3,4. Eine große Herausforderung in MS-basierte Protein Quantifizierung liegt in der Erkennung von niedrig-Fülle Proteinen in komplexen Proben bei ng/mL oder Pg/mL Konzentrationen in Anwesenheit hoher Fülle Proteine und viele Kandidaten-Protein-Biomarkern im menschlichen Plasma gefunden werden innerhalb dieser Reihe5. Dieses Problem wird weitgehend durch die von Natur aus hohen Dynamikbereich und Komplexität der menschlichen Proteoms6verursacht.
Um diese Erkennung Herausforderungen zu überwinden, Immuno-MS-Methoden wurden entwickelt, um bereichern die Zielproteine oder Peptide aus den Musterlösungen auf einer festen Oberfläche, gefolgt von Elution von Analyten und MS Messung7,8 , 9 , 10. durch Immuno-Anreicherung des Analyten werden gereinigt, von komplexen Proben und minimiert die Ion-Unterdrückung Effekte aus anderen Molekülen. Unter den verschiedenen festen Trägern sind magnetische Beads derzeit am häufigsten verwendet, da sie die Vorteile der hohen Antikörper-Bindungskapazität und einfache Handhabung. Magnetische Beads mit unterschiedlichen Functionalizations und Größen wurden entwickelt und vermarktet für Immunopräzipitation Experimente. Bis heute hat Immuno-Anreicherung auf Perlen mit Electrospray Ionisierung (ESI) und MALDI-MS für Proteine und Peptide Messung angeschlossen. In stabiler Isotope Standards und Gefangennahme durch Anti-Peptid-Antikörper (SISCAPA)-Technologie sind Proteine in den Proben verdaut, gefolgt von Inkubation mit Antikörper-beschichtete Perlen für Immuno-Anreicherung. Im "klassischen" SISCAPA sind die aufgenommenen Proteotypic Peptide eluiert von Perlen und gemessen durch flüssige Chromatographie-ESI-MS (LC-MS) oder durch direkte Infusion ESI-Multiple Reaktion Monitoring-MS (ESI-MRM-MS)11,12. Immuno-Anreicherung verbessert die MRM-Assay-Empfindlichkeit um Größenordnungen 3-4 niedrige ng/mL Palette13zu erreichen.
Im Vergleich zu Elektrospray-MS, MALDI-MS ist schneller und beinhaltet jedoch nicht die Reinigung und erneute Gleichgewichtherstellung LC Spalten so gibt es keine Verschleppung und Verschmutzung Probleme eignet sich mehr für Hochdurchsatz-Studien14. Immuno-MALDI-Technologie entwickelt wurde, in unserem Labor, Immuno-Anreicherung mit MALDI-MS für empfindliche und spezifische Quantifizierung von Peptiden und Proteinen (basierend auf Quantifizierung von Proteotypic Peptiden) verbinden15,16 ,17. Nach der Immuno-Bereicherung die Perlen sind auf einem MALDI-Zieltafel hinterlegt, die Matrixlösung wird hinzugefügt, um Perlen und die Platte ist bereit für die Analyse von MALDI-TOF-MS nach dem Trocknen. Elution der Peptide aus den Perlen ist nicht als separater Schritt durchgeführt, aber Affinität-gebundenen Analyten sind durch die MALDI-Matrix-Lösung eluiert, wenn es die Perle Spots, dadurch vereinfachen die Probenvorbereitung und Minimierung Probenverlust hinzugefügt wird. Die iMALDI-Technologie in einer Vielzahl von Anwendungen18,19, angewendet wurde und vor kurzem wurde automatisiert und verwendet für die Messung von Angiotensin ich (Ang ich) zur Bestimmung der Plasma-Renin-Aktivität (PRA)-20. Dieses Protokoll zeigen das Verfahren verwendet, um eine automatisierte iMALDI Assay durchzuführen. Nehmen wir als Beispiel, Inter Tag CVs von weniger als 10 % des PRA-Tests durch Automatisierung, mit der Fähigkeit 744 Proben pro Tag20erreicht.
Der iMALDI PRA-Test zeigte in diesem Manuskript erfordert keine Proteinverdauung, als dem Zielmolekül (Ang ich) ist ein Peptid mit einem Molekulargewicht von 1295.7 Da. In anderen Tests wo Proteinverdauung ist notwendig und eine Peptid dient als Leihmutter für das intakte Protein sollte das ausgewählte Peptid für iMALDI, einzigartig und speziell für das Zielprotein, mit einer Masse von über 800 Da, so daß es leicht aus dem c unterschieden werden können hemical Lärm aus der MALDI-Matrix-Lösung. Anti-Peptid-Antikörper sind erforderlich für die Immuno-Bereicherung der Peptide. Das Protokoll für eine iMALDI-Assay PRA Messung besteht aus vier Schritten: 1) Generation von Ang ich im menschlichen Plasma; (2) Immuno-Anreicherung von Ang ich mit Antikörper-beschichtete Perlen; (3) Übertragung von Perlen zu einem MALDI Zieltafel und Hinzufügen von Matrixlösung; und 4) Erwerb von MALDI-TOF-MS und Daten Analyse20zu signalisieren.

<table width="892">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Healthy control human plasma</td>
			<td class="xl63" style="width:134pt" width="178">Bioreclamation</td>
			<td class="xl68" style="width:89pt" width="119">HMPLEDTA2</td>
			<td class="xl66" style="width:242pt" width="323"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium bicarbonate</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl69">09830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium citrate dibasic</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">09833</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">CHAPS (&#62;=98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">C9426</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Albumin from chicken egg white (&#62;98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">A5503</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ethylenediaminetetraacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">EDS</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Alpha-cyano-4-hydroxycinnamic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">70990</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phosphate buffered saline</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">P4417</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phenylmethanesulfonyl fluoride</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">78830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Trifluoroacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">85172</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetonitrile</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">34967</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">water</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">39253</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">320099</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tris(hydroxymethyl)aminomethane</td>
			<td class="xl63" style="width:134pt" width="178">Roche Diagnostics</td>
			<td class="xl71">3118169001</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Dynabeads Protein G magnetic beads</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">10003D</td>
			<td class="xl66">2.8 &#956;m, 30 mg/mL</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">anti-Ang I goat polyclonal antibody</td>
			<td class="xl63" style="width:134pt" width="178">Santa Cruz Biotechnology</td>
			<td class="xl68" style="width:89pt" width="119">sc-7419</td>
			<td class="xl66"></td>
		</tr>
		<tr height="84" style="height:63.0pt">
			<td class="xl63" height="84" style="width:204pt;height:63.0pt" width="272">Nat and SIS Ang I</td>
			<td class="xl63" style="width:134pt" width="178">synthesized at the University of Victoria-Genome BC Proteomics Centre</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Automated liquid handling system</td>
			<td class="xl63" style="width:134pt" width="178">Agilent</td>
			<td class="xl68" style="width:89pt" width="119">16050-102</td>
			<td class="xl66">Agilent Bravo robotic workstation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12321D</td>
			<td class="xl66">Invitrogen DynaMag-2 magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tube rotator</td>
			<td class="xl63" style="width:134pt" width="178">Theromo Scientific</td>
			<td class="xl68" style="width:89pt" width="119">400110Q</td>
			<td class="xl66">Labquake Tube Rotator</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12027</td>
			<td class="xl66">DynaMag-96 side skirted magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">VP Scientific</td>
			<td class="xl68" style="width:89pt" width="119">771RM-1</td>
			<td class="xl66">used to pull the beads to the bottom of the well</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">MALDI-TOF</td>
			<td class="xl63" style="width:134pt" width="178">Bruker</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66">Bruker Microflex LRF instrument</td>
		</tr>
	</tbody>
</table>

peptide and protein quantification using automated immuno-maldi (imaldi)

Massenspektrometrie (MS) ist eine der am häufigsten verwendeten Technologien zur Quantifizierung von Proteinen in komplexen Proben mit ausgezeichneten Assay Spezifität durch die unmittelbare Erfassung des Masse-Ladungs-Verhältnisses der einzelnen Zielmolekül. Aber MS-basierte Proteomics, wie die meisten anderen analytischen Techniken, hat eine Tendenz zur Messung hoher Fülle Analyten, so dass es schwierig ist, um zu erreichen Nachweisgrenzen von niedrigen ng/mL oder Pg/mL in komplexen Proben, und dies ist der Konzentrationsbereich für viele krankheitsrelevante Proteine in auszahlt wie menschlichen Plasma. Zur Unterstützung bei der Erkennung von niedrig-Fülle Analyten wurde Immuno-Anreicherung integriert in den Assay zu konzentrieren und Reinigen des Analyten vor der MS Messung Assay Empfindlichkeit deutlich zu verbessern. In dieser Arbeit ist die Immuno - Matrix Laser Desorption/Ionisierung (iMALDI)-Technologie präsentiert für die Quantifizierung von Proteinen und Peptiden in auszahlt, basierend auf Immuno-Anreicherung auf Perlen, gefolgt von MALDI-MS Messung ohne vorherige Elution. Die Anti-Peptid-Antikörper sind auf magnetische Beads funktionalisiert und mit Proben inkubiert. Nach dem Waschen, die Perlen sind auf ein MALDI-Zieltafel direkt übertragen und die Signale durch eine MALDI-Zeit des Fluges (MALDI-TOF) Instrument gemessen werden, nachdem die Matrixlösung auf die Perlen angewendet wurde. Die Beispielprozedur Vorbereitung ist im Vergleich zu anderen Immuno-MS-Assays vereinfacht, und die MALDI-Messung ist schnell. Die gesamte Probenvorbereitung ist mit einer Flüssigkeit, die handling-System, mit verbesserten Assay Reproduzierbarkeit und höheren Durchsatz automatisiert. In diesem Artikel dient der iMALDI-Test zur Bestimmung der Peptid Angiotensin ich (Ang ich) Konzentration im Plasma, die klinisch als Auslesen der Plasma-Renin-Aktivität für das Screening von primären Aldosteronism (PA) verwendet wird.

Eine automatisierte iMALDI-Verfahren zur Messung der Ang ist mir in Abbildung 1gezeigt. Ziel-Peptide (endogene Peptide oder Peptide aus verdauliche Proteine) sind auf Anti-Peptid magnetische Beads bereichert, und dann werden die Perlen auf eine Zieltafel für MALDI-Messung übertragen. Das gesamte Verfahren wird vereinfacht, im Vergleich zu anderen Immuno-MS-Technologien, die zusätzliche Peptid Elution Schritte erfordern. Automatisierung des iMALDI-Tests kan...

Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Ein Protokoll für die Quantifizierung von Proteinen in komplexen biologischen Flüssigkeiten mit automatisierten Immuno-MALDI (iMALDI)-Technologie präsentiert.

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 ...

peptide-and-protein-quantification-using-automated-immuno-maldi-imaldi

Research

JoVE Journal

Chemistry

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

7.9K Aufrufe.  University of Victoria-Genome BC Proteomics Centre. Ein Protokoll für die Quantifizierung von Proteinen in komplexen biologischen Flüssigkeiten mit automatisierten Immuno-MALDI (iMALDI)-Technologie präsentiert.

Serie: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Produktion von Disulfid-stabilisierte Transmembrane Peptid-Komplexe für Structural Studies

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Forschung

Chemie

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Multiplex-Fluoreszenz-Mikroarray für Menschenspeichelproteinanalytik Verwendung von Polymer-Mikrosphären und LWL-Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

Analyse von Proteindynamik mit Wasserstoffwechsel Massenspektrometrie

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-Pool-Synthese und Charakterisierung von Peptid-Amid-Tertiär-Bibliothek

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Entwicklung eines Backbone zyklische Peptid-Bibliothek als potentielle Antiparasiten Therapeutics unter Mikrowellenbestrahlung

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

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

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE - Clear india geleluiert flüssige Fraktion Verleitung Elektrophorese

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

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Charakterisierung, Quantifizierung und substanzspezifische Isotopenanalyse von Pyrogene Kohle Benzolpolycarbonsäuren (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Kapillarelektrophorese zum Monitor Peptid Verpflanzen auf Chitosanfilme in Echtzeit

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

In Situ Erkennung und Quantifizierung der einzelnen Zelle von Metall-Oxid-Nanopartikel mit nuklearen Mikrosonde Analyse

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...