Diese Arbeit wird teilweise von den National Institutes of Health Grants GM-26738 (an JAT) unterstützt.

1. PAK2 Activation <ol><li> Fügen Sie die entsprechenden vorgeprüft Höhe von Caspase 3 bis 20 ul von 12 mg / ml PAK2 in Puffer A (50 mM Tris-HCl, 150 mM NaCl und 2 mM DTT bei pH 7,5) und Inkubieren bei 34 ° C für 30 min vollständig zu spalten und zu aktivieren PAK2. </li><li> Analysieren Sie die Spaltprodukte durch SDS-PAGE (10%) und Coommassie-Blau-Färbung. </li><li> Fügen Sie die gespalten PAK2 zur Aktivierung Puffer B (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl 2, 10 mM ATP) bei pH 7,5, in 20 ul bis zu einer Endkonzentration von 10 mg / ml PAK2, und inkubieren bei 34 ° C für 30 min vollständig zu aktivieren PAK2 durch Autophosphorylierung an 8 Standorten. </li><li> Bestätigen Sie volle Spaltung PAK2 durch den Zuzug von p34 und p27-Fragmente durch SDS-PAGE 10 bestimmt. Proper Überprüfung der Autophosphorylierung Bedingungen über Autoradiographie in einem separaten Experiment mit (32 P) ATP oder Massenspektrometrie Phosphopeptide erkennen hergestellt. </li></ol> 2. Identifikationvon Pepsin verdaut PAK2 Fragments <ol><li> 1 ml 0,1% Trifluoressigsäure (TFA) bei pH 2,5, die Agaroseperlen zweimal vor waschen zu verwenden. Dann Zentrifuge die Proben für 15 sec bei 2.000 xg, um die Perlen zu entfernen. </li><li> Add PAK2 (20 ug in 2 ul 150 mM NaCl, 50 mM Tris-HCl und 2 mM DTT bei pH 7,5) auf 100 ul 0,1% TFA, und inkubieren Sie die Probe für 5 min mit 30 ul Pepsin-konjugierten Agaroseperlen . </li><li> Äquilibrieren einer Reverse-Phase-HPLC-C18-Säule mit einem primären Lösungsmittel-System von 0,1% TFA bei pH 2,5. Das Pepsin verdaut (50 ul) wird eluiert mit einem linearen 100-min Steigung von 0 bis 80% Acetonitril mit 0,1% TFA bei einer Fließgeschwindigkeit von 0,2 ml / min. Die Fraktionen werden alle 2 min gesammelt. </li><li> Trocknen Sie die HPLC-Fraktionen mit einem Speed-Vac (3 h) und Resuspendieren der Proben in eine Lösung von Acetonitril (5 ul) und 0,1% TFA (5 ul) bei pH 2,5. </li><li> Zunächst wird jede Fraktion für Peptide mit der MALDI-TOF PerSeptive Biosystems Voyager DE STR (PE Bi abgeschirmtosystems, Foster City, CA, USA). </li><li> Die 16 Fraktionen, die Peptide sind Tandem-Massenspektrometrie mit einem Q-TOF-Massenspektrometer und ein QStar XL oMALDI MS / MS, um die Fragmente zu identifizieren unterzogen. </li><li> Die Abfolge der einzelnen Peptid wird von der Produkt-Ionen durch die Tandem-Massenspektrometrie mit den theoretischen m / z-Verhältnisse auf dem Primär-Sequenz von PAK2 mit dem Protein Prospector Website (basierend generiert überprüft <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Messung von Amid H / D-Austausch <ol><li> Halten Sie alle Lösungen und Reagenzien für die H / D-Austausch bei 25 ° C benötigt in einem Wasserbad. ATP wird in Wasser gelöst und der pH-Wert auf 7,0 vor der Anwendung angepasst. Die zusätzliche 4mm ATP in die D 2 O-Puffer ist ATP dissoziiert verhindern PAK2 nach Verdünnung. </li><li> Initiate H / D-Austausch durch Zugabe von 18 ul der gepufferten D 2 O (50 mM MOPS pH 6.98, 125 mM NaCl) bis 2 ul (20 ug) Von PAK2 in einem Puffer mit 150 mM NaCl, 50 mM Tris-HCl (pH 7,5) und 2 mM DTT, was zu einer PAK2 von 10 mg / ml und einem pH-Wert von 7,0 bei 0 ° C. </li><li> Inkubieren Sie die Proben in dreifacher Ausfertigung in der H / D-Austausch für 0, 0,5, 1, 3, 5 und 10 min bzw. 24 h. </li><li> Quench der H / D-Austausch-Prozess durch Zugabe von 180 ul eiskaltem 0,1% TFA bei pH 2,2, die den pH-Wert bringt bis 2,5 in einem Gesamtvolumen von 200 ul. Für die Proben mit ATP, 180 ul eiskalter 0,11% TFA bei pH 2,2 wird verwendet, um den pH-Wert bei 2,5 im vergüteten Zustand zu halten. </li><li> Fügen Sie ein Aliquot (100 ul) jeder abgeschreckten Probe zu 30 ul aktiviert Pepsin-konjugierten Agaroseperlen. Die Pepsinverdau darf auf Eis für fünf Minuten gehen und die Proben werden alle 30 sec gevortext. </li><li> Beenden Sie die Verdauung durch Zentrifugation der Probe für 15 sec bei 5.000 xg bei 4 ° C zur Entfernung des Pepsins. </li><li> Schnell einfrieren der Proben in flüssigem N 2 und bei -80 ° C für nicht mehr als 2Tage vor der MALDI-TOF-Analyse. </li><li> Die Matrix für MALDI beträgt 5 mg / ml α-cyano-4-Hydroxy-Säure (CHCA) in einer Lösung (1:1:1) Acetonitril, Ethanol und 0,1% TFA bei pH 2,5 aufgelöst und statt bei 0 ° C. </li><li> Teilweise Tauwetter jede Probe schnell und mischen Sie 1 ul jeder Probe mit 1 ul der Matrix und der Fleck auf 4 ° C MALDI Zieltafel. </li><li> Tragen Sie eine moderate Vakuum auf die Platte, um die Stellen in 30 sec vor der MALDI-TOF-Analyse trocken. Die Zeit zwischen dem Auftauen der Probe und Spektrum Abruf ist ca. 3 min. </li><li> Erwerben Sie MALDI-TOF-Massenspektren mit einem PerSeptive Biosystems Voyager DE STR. Erfassung von Daten in einem 2-GHz Abtastrate bei 100.000 Datenkanäle mit einem 20.000-V Beschleunigungsspannung und eine 65% Netzspannung mit verzögerter Extraktion mit einem 180-ns-Impuls zu verzögern. Einhundert-Scans sind in 2 min gemittelt. Die MALDI-TOF Instrument ist nicht auf eine Marke oder eine bestimmte Software beschränkt. </li><li> Verwenden Sie die Data Explorer 4.0 Computer-Programm zu calculaß die Deuterierung von jedem Fragment. </li><li> Berechnen Sie die Rückseite Austausch über das Verhältnis der tatsächlichen eingebaut Deuteron Zahl bei 24 Stunden von H / D-Austausch mit allen möglichen austauschbaren Stellen, die das Rückgrat Amiden mit Ausnahme des N-terminalen Amid (Abbildung 1) dividiert wird. </li></ol> 4. Repräsentative Ergebnisse: Die Caspase-Spaltung und Autophosphorylierung durch SDS-PAGE Commassie Färbung überprüft. Inaktive PAK2 ist eine einzelne Bande von 58 kDa. Caspase Spaltung PAK2 abgeschlossen ist, und erzeugt zwei Fragmente, p27 und p34, die separat zu migrieren. Die Migration von autophosphorylierten p27 und p34 zeigt eine Überlappung auf das Gel aufgrund der verzögerten Migration der vollständig phosphoryliert p27-Fragment (Abbildung 2). H / D-Austausch Experimente werden mehrmals zwischen 0 und 10 min (Abbildung 3) durchgeführt. Als Beispiel den zeitlichen Verlauf von Deuterium Einbindung in die m / zpeak 1697,85 inaktiver PAK2 besteht aus mehreren Isotopen Gipfel als durch die Verschiebung der Masse Umschlag im Laufe der Zeit gezeigt, zusammen. <img alt="Abbildung 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Abbildung 1. Gleichung für die Berechnung der Deuterierung. <img alt="Abbildung 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Abbildung 2. Autophosphorylierung und Caspase Spaltung PAK2. Caspase-abgespalten PAK2 (linke Spur), intakte inaktiv PAK2 (mittlere Spur) und Caspase-abgespalten autophosphorylierten PAK2 (rechte Spur) wurden durch SDS-PAGE und Coommassie-Blau-Färbung analysiert. Angepasst von Hsu, YH et al 2. <img alt="Abbildung 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Abbildung 3. Massenspektrum einer zeitlichen Verlauf der Deuterium-Einbau für inaktive PAK2. Inaktive PAK2 wurde H / D excha unterzogennge für 0-10 min und mittels MALDI-TOF. Ein Beispiel für die erweiterte Isotopenverteilung von MALDI-TOF von Peak m / z 1697,85 während der zeitliche Verlauf der H / D-Austausch wird angezeigt. Die rote gestrichelte Linie zeigt den Durchschnitt der Masse Umschlag und die Verschiebung der Masse Umschlag zeigt die Deuterierung eines Peptids.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, 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=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

Keine Interessenskonflikte erklärt.

Bezeichnung des Pepsin-verdauten Fragmente ist ein kritischer Schritt der H / D-Austausch zu experimentieren. Eine falsche Identifizierung des Peptids kann zu einer falschen Schlussfolgerung führen. Pepsin verdaut PAK2 wird durch einen Reverse-Phase-HPLC-C18-Säule eluiert, um einen sauberen Hintergrund zu erhalten. In unseren Experimenten wurden insgesamt 40 Fraktionen gesammelt und einer MALDI-TOF. Multiple Peptide könnten in jeder der Fraktionen identifiziert werden. Die Peptide wurden auf Tandem-Massenspektrometrie (Q-TOF MS / MS und oMALDI MS / MS) unterzogen, um ihre Sequenzen zu identifizieren. Die MS / MS-Spektren eines Peptids sollte mindestens drei Produkt-Ionen, um die Identifizierung des Peptids zu bestätigen. Der Data Explorer 4.0-Computer-Programm (Applied Biosystems) führt die anfängliche Basislinienkorrektur und Lärm Filtration. Jedes Spektrum ist auf zwei sequenziert Gipfel kalibriert werden. Wir kalibrieren unser Spektrum von der theoretischen Masse des undeuterierten m / z, die 923,45 und 1697,84 sind. Ter Massengenauigkeit nach der Kalibrierung erreichen 10 ppm. Nach Deuterierung kann der Mono-Isotop eine höhere Masse zu verlagern. Unitary Schritt erhöht, um diese m / z-Verhältnissen ergab die verbesserte Massen mit Deuterierung verbunden. Die Peak-Intensitäten der Masse Umschlag wird keinen Einfluss auf die Deuterierung Ebene. Allerdings sind die Peak-Intensitäten der Isotopen-Peaks in einem bestimmten Masse Umschlag von entscheidender Bedeutung für die Berechnung der durchschnittlichen Masse. Der erste Schritt der Berechnung der eingelagerten Deuteronen ist Subtraktion der peptide mass der nicht-deuterierten Probe aus der deuterierten Probe. Drei weitere Faktoren, die D 2 O verdünnt, die restliche Deuteronen in den Seitenketten und der Rückseite Austausch, müssen weiter in der Berechnung berücksichtigt werden (Abbildung 1). Die D 2 O ist die Verdünnung Faktor D 2 O nach dem Mischen der Probe und D 2 O-Puffer auf die H / D-Austausch zu initiieren. Die verbleibende Deuteron (4,5%) in den Seitenketten der Pepsin-Verdauunged Peptide muss abgezogen werden. Die Back-Austausch ist der unvermeidliche Verlust von Deuterierung in allen deuterierten und Pepsin verdauten Peptide in der Masse Messvorgang. Die meisten Lösungsmittel zugänglich Peptid (m / z = 1105,60) nach 24-stündiger der H / D-Austausch stellt eine vollständige Deuterierung Region. Die eingearbeiteten Deuteron-Nummer für jedes der Peptide ist der Unterschied zwischen dem Schwerpunkt des deuterierten und nicht deuterierten PAK2 peptischen Peptide. Die am höchsten deuterierten Peptid m / z 1105 wurde ausgewählt, um volle Deuterierung stellen, wenn PAK2 wurde bei 24 Stunden von H / D-Austausch. The Back Exchange Ratio wurde von der tatsächlichen eingebaut Deuteron Zahl bei 24 Stunden von H / D-Austausch mit allen möglichen austauschbaren Stellen im Peptid m / z 1105 dividiert wird.

<table border="1"></table><table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-cyano-4-Hydroxy-Säure </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager DE STR </td><td></td></tr><tr><td> Q-TOF-Massenspektrometer </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QStar XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Data Explorer 4.0-Computer-Programm </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

Amid Wasserstoff / Deuterium-Austausch (H / D-Austausch) gekoppelt mit der Massenspektrometrie wurde in großem Umfang verwendet werden, um die Schnittstelle von Protein-Protein-Interaktionen, Protein Konformationsänderungen, Protein-Dynamik und Protein-Ligand-Interaktionen zu analysieren. H / D-Austausch auf der Amid-Positionen wurde verwendet, um die Deuterierung Preise der Mikro-Regionen in einem Protein durch Massenspektrometrie 1,2,3 messen. Die Auflösung dieser Methode hängt von Pepsinverdau der deuterierten Protein von Interesse in Peptide, die normalerweise im Bereich von 3 bis 20 Resten. Obwohl die Auflösung des H / D-Austausch durch Massenspektrometrie gemessen niedriger ist als die eines einzigen Rückstands Beschluss der Heteronucleare Single Quantum Coherence (HSQC)-Methode der NMR, Massenspektrometrie Messung in H / D-Austausch gemessen wird nicht durch die Größe der eingeschränkten protein 4. H / D-Austausch erfolgt in einer wässrigen Lösung, die Proteinkonformation unterhält durchgeführt. Wir bieten Ihnen eine Methode, die utilIZES die MALDI-TOF zur Detektion 2, statt einer HPLC / ESI (Elektrospray-Ionisation)-MS-System 5,6. Die MALDI-TOF liefert genaue Masse Intensität Daten für die Peptide der verdauten Proteins, in diesem Fall Proteinkinase PAK2 (auch als γ-Pak). Proteolyse von Pak 2 ist in einer Offline-Pepsinverdau durchgeführt. Diese alternative Methode, wenn der Benutzer keinen Zugriff auf eine HPLC und Pepsin Säule verbunden mit der Massenspektrometrie, oder wenn das Pepsin Spalte HPLC nicht in eine optimale Verdauung Karte führen, zum Beispiel, die stark Disulfid-gebundenen abgesondert Phospholipase A 2 (SPLA 2). Mit Hilfe dieser Methode haben wir erfolgreich überwacht Veränderungen in der Deuterierung Ebene während der Aktivierung des PAK2 durch Caspase-3 Spaltung und Autophosphorylierung 7,8,9.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI-TOF-Massenspektrometrie wurde erfolgreich genutzt, um die Amid-Wasserstoff / Deuterium-Austausch in Proteinkinase PAK2 Aktivierung überwachen.

Amid Wasserstoff / Deuterium-Austausch &amp; MALDI-TOF-Massenspektrometrie Analyse der PAK2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

amide-hydrogendeuterium-exchange-maldi-tof-mass-spectrometry-analysis

Research

JoVE Journal

Biology

17.7K Aufrufe.  Tunghai University. MALDI-TOF-Massenspektrometrie wurde erfolgreich genutzt, um die Amid-Wasserstoff / Deuterium-Austausch in Proteinkinase PAK2 Aktivierung überwachen.

Serie: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI-Probenvorbereitung: Die Ultra Thin-Layer-Methode

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Forschung

Biologie

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analyse großer Proteinkomplexe von Structural-Massenspektrometrie

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-Wellen-Ionen-Mobilitäts-Massenspektrometrie: Basic Experimentelle Verfahren zur Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Direkte Analyse von Einzelzellen durch Massenspektrometrie bei Atmosphärendruck

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantifizierung von Proteinen über ein Peptid Immunoaffinity Enrichment gekoppelt mit der Massenspektrometrie

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Thiolredox Proteome Mit Isotopen-Massenspektrometrie Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

Die Verwendung von MALDI-TOF-Massenspektrometrie und einer Kundendatenbank zu den Bakterien Indigene auf eine einzigartige Höhle Umwelt (Kartchner Caverns, AZ, USA) Charakterisieren

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Selected Reaction Monitoring-Massenspektrometrie für Absolute Proteinquantifizierung

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Anreicherung von extrazellulären Matrixproteinen aus Gewebe und Verdauung in Peptide für Massenspektrometrie-Analyse

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Anwendungen der Einzel-Sonde: Mass Spectrometry Imaging und Einzelzellanalyse unter Umgebungsbedingungen

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...