Diese Arbeit wurde von der NCI Clinical Proteomics Technology Assessment Center (CPTAC) zu gewähren (# U24 CA126476) sowie einen Zuschuss von der Entertainment Industry Foundation (EIF) und der EIF Frauen Cancer Research Fund der Breast Cancer Biomarker Discovery Consortium finanziert und großzügige Geschenke von der Keck Foundation, die Kanarischen Stiftung und der Paul G. Allen Family Foundation.

Versuchsdurchführung: Der Test erfordert synthetische Peptide und anti-Peptid-Antikörper. Ausgewählte Peptide sollte eindeutig sein, um das Protein von Interesse, enthalten zwischen 8 und 22 Aminosäuren, und haben keine bekannten post-translationale Modifikationen. Methionin-Reste sind in der Regel vermieden und Peptide, die zweibasische Aminosäuren (zB KK, KR, RR) sind unerwünscht. Bei dieser Technik ist es üblich, stabiles Isotop markierten Peptiden als interne Standards zu verwenden, vereinigt Hochleistungs (13 C und 15 N) markierten Aminosäuren in der C-Terminus des Peptids (dh K oder R gekennzeichnet). Das folgende Protokoll beschreibt ein Assay entwickelt, um das Peptid GDSLAYGLR von der Maus-Protein Osteopontin messen, mit anti-Peptid-Antikörper aus Epitomics Inc. (Burlingame, CA) und synthetischer Peptide, die aus New England Peptide (Gardner, MA) erhalten. Das Protokoll besteht aus drei Schritten (Abbildung 1): 1) Trypsin Verdauung der komplexen Proteingemisch, 2) Anreicherung von Peptiden 3) Analyse mittels Massenspektrometrie. Es wird auf einer menschlichen Plasmaprobe mit der Maus Osteopontin Protein versetzt nachgewiesen werden. 1. Trypsin enzymatische Verdauung und Bereinigung <ol><li> Tauwetter 10 ul ordentlich Plasma Aliquot auf nassem Eis. </li><li> Bestimmen Sie die Gesamt-Protein-Konzentration von BCA-Assay und Zentrifuge die Probe auf eine Entfernung von Schwebstoffen. </li><li> Pipettieren 10 l Aliquot von seiner Lagerung Rohr zu 1000 ul Deepwell Platte und Cover mit Pierce-fähig Film. </li><li> Fügen Sie 20 ml frisches 9M Harnstoff / 30mm Dithiothreitol (DTT) (Endkonzentration 6M Harnstoff / 20mm DTT) zu jeder Probe. </li><li> Inkubieren für 30 Minuten bei 37 ° C. </li><li> Add 3 ml frisches 500 mM iodoacetamide (final IAM 50 mM) zu jeder Probe. </li><li> Inkubieren für 30 Minuten im Dunkeln bei Raumtemperatur. </li><li> Add 257 ul 100 mM Tris (pH 8) (verwässert Harnstoff auf ~ 0,6 Mio.). </li><li> Add 10 l Trypsin-Stammlösung (1 ug / ul; für 01.50 Enzym: Substrat-Verhältnis). </li><li> Inkubieren bei 37 ° C über Nacht (12-16 Stunden). </li><li> Add 3 ul von tausenden von Ameisensäure (Endkonzentration 1%). </li><li> Add stabilen Isotopen-Standard (multiple Standards hinzugefügt werden, wenn der Durchführung einer Multiplex-Assays, in der Regel ist dies etwa 10 ul mit 50-100 fmol von Standard-markiertem Peptid). </li><li> Waschen Sie die Patrone Oasis Platte auch mit 500 ul 0,1% Ameisensäure in 80% Acetonitril, das Verwerfen der Durchströmung. Wiederholen Sie diese 3-fache. </li><li> Äquilibrieren Sie die Patrone Platte durch Zugabe von 500 ul von 0,1% Ameisensäure in Wasser, und entsorgen Sie die Flow-Through. Wiederholen Sie diese 4-fache. </li><li> Legen Sie verdauen Proben, um die Patrone Platte und stellen Sie das Vakuum, so dass die Strömung ist sehr langsam. </li><li> Wash mit 500 ul 0,1% Ameisensäure in Wasser, und entsorgen Sie Flow-Through. Wiederholen Sie diese 3-fache. </li><li> Eluieren Peptide durch Zugabe von 2 x 500 ul von 0,1% Ameisensäure in 80% Acetonitril in 1000 ul Deep-Well-Platte (nicht verwerfen flow-through). </li><li> Lyophilisieren (oder Speedvac) das Eluat. (Gefriertrocknung ist die bevorzugte Methode) </li><li> Rekonstituieren getrockneten Peptide durch Zugabe von 50 ul PBS + 0,03% CHAPS. </li></ol> 2. Peptide Immunaffinitäts Bereicherung <ol><li> Übertragen Sie die Probe auf Standard Kingfisher 96 Well-Platten. </li><li> Add 1 pg Antikörper und 1,5 ul Protein-G-beschichteten magnetischen Kügelchen pro Ziel (es ist optional an den Antikörper an die Perlen vor der Zugabe zu vernetzen). Stellen Sie sicher, Perlen sind auch durch Schütteln oder Vortexen suspendiert. </li><li> Die Platte mit Folie. </li><li> Inkubieren über Nacht (12-16 Stunden) mit sanften Taumeln, um sicherzustellen, Perlen aufgehängt sind. </li><li> Centrifuge Platte bei 32 xg für 5 Sekunden, um eine Flüssigkeit aus der Folie Oberfläche zu entfernen. </li><li> Entfernen Sie die Folie abdecken. </li><li> Waschen und Elution der Perlen kann manuell oder in einer automatisierten Weise durchgeführt werden. Dieses Verfahren beschreibt die Schritte auf einem Kingfisher-Plattform automatisiert. </li><li> Waschen Sie die Kugeln 2 mal mit 200 ul PBS + 0,03% CHAPS (1 Minute pro Waschgang). </li><li> Waschen Sie die Kugeln 1 mal mit 200 ul 1:10 Verdünnung des PBS + 0,03% CHAPS (1 Minute). </li><li> Die Peptide werden in 25 uL von 5% Essigsäure + 0,03% CHAPS eluiert. </li><li> Setzen Sie den Elutions-Platte auf einem Magneten und Transfer das Eluat einer 96-Well-Platte, die Pflege nicht auf die übrig gebliebenen Perlen zu übertragen. </li><li> Die Platte mit einem Dichtmatte. </li><li> Die Platte enthält das Eluat ist auf den dreifachen quadrapole Massenspektrometer für die Analyse geliefert. </li></ol> 3. Die Analyse durch multiple reaction monitoring - Massenspektrometrie <ol><li> Transitions für SRM / MRM Analyse ausgewählt und durch Infusion einer 1 Picogramm pro Mikroliter Lösung von Peptid-Standard in 30% acetonitrile/0.1% Ameisensäure in das Massenspektrometer bei 0,5 ul / min optimiert werden. Sobald stabile Spray erhalten wird, sammelt ein MS / MS-Spektrum. </li><li> Übergänge sind von der MS / MS-Spektrum durch die Identifizierung thre ausgewählte reichlich Fragment-Ionen in einem Bereich des Spektrums mit wenig Lärm. Für mehrfach geladenen Peptid-Ionen, die y-Ionen-Fragmente bei m / z erkannt&gt; Vorläufer m / z sind in der Regel das Beste für SRM / MRM Assay-Entwicklung. Abbildung 2 zeigt die MS / MS-Spektrum und ausgewählte Übergang Ionen 476,3&gt; 508,3, 579,3 , 692,4 (Übergänge für die schweren stabilen Isotopen Standard 481,3&gt; 518,3, 589,3, 702,4 nicht dargestellt) für das Peptid GDSLAYGLR. </li><li> Je nach Marke und Modell des Massenspektrometers verwendet, gibt es mehrere Parameter, die für jeden Übergang optimiert werden kann. Hier optimierten wir die Aufprallenergie der jeweils ausgewählten Übergang von Rampen die Aufprallenergie und Überwachung der Pegel des Signals. Eine Kollision Energie von 25 wurde für jeden Übergang verwendet. </li><li> Kurz gesagt, ist eine typische Konfiguration für SRM / MRM Analyse wie folgt: Mobile Phase (A) 0,1% Ameisensäure, (B) 90% Acetonitril / 0,1% Ameisensäure, 0,3 x 5 mm C18 Trap-Säule, 75 um ID x 15 cm C18 Trennsäule (Reprosil-Pur C18 AQ, 120 A ° Pore). Das Injektionsvolumen beträgt 10 ul und die Probe für 5 min bei 3% B bei einer Gesamt-Volumenstrom geladen 3 ul / min und eluiert mit einem linearen Gradienten von 3 bis 45% B bei 300 bis 400 nL / min in 10 min. Bedingungen auf einem 4000 QTRAP (ABSCIEX, Foster City, CA) wurden spray Spannung 2.3kV, Ionenquelle 150 ° C, GS1 von 12 und Vorhang Gas 15. </li><li> Die Probe wird durch SRM / MRM durch die Injektion von 10 ul der Probe analysiert. Die Peakflächen sind für leichte und schwere Peptide mit Hilfe des Programms Skyline integriert. 14 Berechnen Sie die Peakflächenverhältnis (PAR) von unmarkiertem / markiertes Peptid in jeder Probe unter Verwendung der am häufigsten vorkommenden Übergang, der frei von Nebengeräuschen ist, in diesem Fall die y5 Übergang (Licht-Peptid 476,3&gt; 579,3, schwere Standard-Peptid 481,3&gt; 589,3). </li></ol> 4. Repräsentative Ergebnisse: Gemessen Peakflächenverhältnisse (light endogenen Peptids gegenüber versetzt schweren Isotopen-markiertes Peptid) liefern ein quantitatives Maß für die Ziel-Peptid. Abbildung 3 zeigt ein Beispiel-Chromatogramm von leichten und schweren Peptide in einem SISCAPA angereicherten Probe. Beachten Sie, dass leichte und schwere Peptide eluieren gleichzeitig und mehrere Übergänge für jedes Peptid, um die Identität bestätigen können überwacht werden. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Eine schematische Übersicht über die SISCAPA Prozess. Eine komplexe Proteingemisch wird in Peptide verdaut. Gezielte Peptid Analyten (endogenen Analyten und einer Stachelwalze stabile Isotop-markierten internen Standard) angereichert mit anti-Peptid-Antikörper auf Protein G-beschichteten magnetischen Partikeln immobilisiert. Nach der Isolierung sind die gezielte Peptide, die aus den magnetischen Partikeln eluiert und massenspektrometrisch analysiert zur Quantifizierung in Bezug auf den internen Standard. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Abbildung 2"/> Abbildung 2. MS / MS-Spektrum des Peptids GDSLAYGLR zeigt Auswahl von drei Fragmente für SRM / MRM-Übergänge. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Beispiel Chromatogramme zeigen die Peak-Profil eines Licht-Peptid Analyten (rot) und der schweren stabilen Isotopen-markierten internen Standard (blau). Chromatogramme für die y5 Übergang von der Licht-Peptid (476,3&gt; 579,3) und schwere stabile Isotop markiert Standard (481,3&gt; 589,3) sind über der Zeit aufgetragen, wie sie aus der Chromatographie-System zu eluieren.

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Der wichtigste Schritt in das Protokoll wie oben beschrieben ist die Gewährleistung der Perlen bleiben gut gemischten während der Inkubationszeit. Erlauben die Perlen auf den Grund des Brunnens / Fläschchen niederlassen wird in erhöhte Variabilität führen. Es ist auch wichtig, Spin-down die Flüssigkeit ab, auf der Spitze des Brunnens / Fläschchen nach der Inkubationszeit bleiben kann. Reproduzierbare Trypsinverdauung ist ebenfalls entscheidend. Das Verfahren für die Verdauung hier beschriebenen wurde ausgiebig in unserem Labor in Verbindung mit SISCAPA eingesetzt, jedoch ist es wahrscheinlich, alternative Methoden der Verdauung für eine gegebene Menge von Target-Proteinen konnte optimiert werden 15 Elution der freien Antikörper nicht mit dem Nachweis der stört. Peptid, wahrscheinlich, weil die Antikörper aus der Säule eluiert oder später als die Peptide ist ausgeschlossen, aber die Antikörper gegen das Protein G Kügelchen vernetzt vor Affinitätsanreicherung in großen Experimente oder wenn freie Antikörper werden zum Problem. Wir haben auch festgestellt, es sinnvoll, einen Magneten unterhalb der Probe Platte auf dem Autosampler sowie das Waschen der Trap-Säule in die umgekehrte Richtung des Flusses (im Vergleich zu Be-) Platz, um restliche Perlen oder Partikel zu entfernen. Zusätzlich zu den Magnetic-Bead-Konzept beschrieben, kann die Technik auch, um eine Spalte Format (dh Affinitätschromatographie) angepasst werden. 12, 16 Sobald der Benutzer bequem mit dem Gesamt-Protokoll, ist die Technik auch zugänglich für mehrere Modifikationen, die die gesamte Assay zu verbessern. 11 Zunächst ist es möglich, mehrere Analyten in einem einzigen Test zu analysieren durch die Kombination von Antikörpern in der Anreicherung (dh Multiplexing). Das Massenspektrometer kann die gleichzeitige Analyse einer großen Anzahl von Analyten. Zweitens erhöht das Volumen der ursprünglichen Probe verbessert die Sensitivität des Tests.

<table border="1"><tbody><tr><th> Bezeichnung des Stoffes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> 1000ul Tiefbrunnen 96-Well-Platten </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc Kartusche Platte, Sep-pak, 40 mg 96-Well-Platte </td><td> Waters </td><td> 186003966 </td></tr><tr><td> Kingfisher 96-Well-Platte </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> Klare 96-Well, weiß Wandplatte </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> Folienabdeckung für 96-Well-Platte </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Axymat Dichtmatte für 96-Well-Platte </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X Pierce Film </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher Magnetic-Bead-Prozessor mit PCR Magnetkopf </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tabelle 1: Werkstoffe <table border="1"><tbody><tr><th> Bezeichnung des Stoffes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Harnstoff </td><td> Sigma Aldrich </td><td> U6031 </td><td></td></tr><tr><td> Trizma Basis </td><td> Sigma Aldrich </td><td> T1503 </td><td></td></tr><tr><td> Dithiothreitol (DTT) </td><td> Durchstechen </td><td> 20291 </td><td> &quot;No-wiegen Dithiothreitol&quot; </td></tr><tr><td> Iodacetamid (IAM) </td><td> Sigma Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Trypsin Gold- </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Protein G magnetische Kügelchen, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Phosphat-gepufferte Salzlösung (PBS) </td><td> Thermo Fisher Scientific </td><td> L5401 </td><td></td></tr><tr><td> CHAPS </td><td> Thermo Fisher Scientific </td><td> 28300 </td><td></td></tr><tr><td> Ameisensäure </td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td> Acetonitril </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> Essigsäure </td><td> Sigma Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tabelle 2: Reagenzien <table border="1"><tbody><tr><th> Lösung </th><th> Kommentare (optional) </th></tr><tr><td> 100 mM Tris pH 8 </td><td></td></tr><tr><td> 9 M Harnstoff / 30 mM DTT in 100 mM Tris (pH 8) </td><td> muss jedes Mal frisch zubereitet werden </td></tr><tr><td> 500 mM IAM in 100 mM Tris (pH 8) </td><td> muss jedes Mal frisch zubereitet werden </td></tr><tr><td> Trypsin 1mg/ml in 100 mM Tris pH 8 </td><td></td></tr><tr><td> Trypsin-Inhibitor 1mg/ml in 100 mM Tris pH 8 </td><td></td></tr><tr><td> Stabile Isotope Standard Mastermix </td><td></td></tr><tr><td> Anti-Peptid-Antikörper-Mastermix </td><td></td></tr></tbody></table> Tabelle 3: Lösungen vorbereitet werden

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Es gibt einen großen Bedarf für quantitative Assays in Messung von Proteinen. Traditionelle Sandwich-Immunoassays, vor allem als der Goldstandard in Quantifizierung sind mit hohen Kosten, langen Lieferzeiten verbunden sind, und sind voller Nachteile (zB heterophile Antikörper, Autoantikörper Störungen &quot;hook-Effekt&quot;). 1 Eine alternative Technik ist Affinitätsanreicherung von Peptiden mit quantitativen Massenspektrometrie gekoppelt, die gemeinhin als SISCAPA (Stable Isotope Standards und Capture von Anti-Peptid-Antikörper) bezeichnet. 2 In dieser Technik Affinitätsanreicherung von Peptiden mit Isotopenverdünnungsanalyse und Detektion von ausgewählten / Multiple Reaction Monitoring-Massenspektrometrie ( SRM / MRM-MS) liefert quantitative Messung von Peptiden als Ersatz für ihre jeweiligen Proteine. SRM / MRM-MS gut ist für eine genaue Quantifizierung kleiner Moleküle 3, 4 eingerichtet und in jüngerer Zeit wurde angepasst, um die Konzentrationen von Proteinen in Plasma-und Zell-Lysaten zu messen. 5-7 Zur Quantifizierung von Proteinen zu erreichen, werden diese größeren Moleküle verdaut Komponente Peptide mit Hilfe eines Enzyms wie Trypsin. Ein oder mehrere ausgewählte Peptide, deren Sequenz ist einzigartig an das Zielprotein in dieser Art (zB &quot;proteotypic&quot; Peptide) werden dann aus der Probe mit anti-Peptid-Antikörper angereichert und gemessen als quantitative stöchiometrische Surrogate für Protein-Konzentration in der Probe. Daher, um Isotopenverdünnungsanalyse (SID) Methoden (dh einer Stachelwalze-in stabilen Isotopen markiertes Peptid-Standard), SRM / MRM kann verwendet werden, um Konzentrationen von proteotypic Peptide als Ersatz für die Quantifizierung von Proteinen in komplexen biologischen Matrices messen. Gekoppelte Die Assays haben mehrere Vorteile im Vergleich zu herkömmlichen Immunoassays. Die Reagenzien sind relativ billiger zu erzeugen, die Spezifität für den Analyten ist ausgezeichnet, die Tests sehr gemultiplext werden können, kann eine Anreicherung von ordentlichen Plasma (kein Abbau erforderlich) durchgeführt werden, und die Technik ist offen für ein breites Spektrum von Proteinen oder Modifikationen von Interesse. 8-13 In diesem Video zeigen wir die grundlegende Protokoll als eine Magnetic-Bead-Plattform angepasst.

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Stabile Isotope Standards und Capture von Anti-Peptid-Antikörper (SISCAPA) Paare Affinitätsanreicherung von Peptiden mit Isotopenverdünnungsanalyse Massenspektrometrie (MRM-MS) zur quantitativen Messung von Peptiden als Ersatz für ihre jeweiligen Proteine ​​liefern. Hier beschreiben wir das Protokoll mit magnetischen Partikeln in einer teilautomatisierten 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 ...

quantification-proteins-using-peptide-immunoaffinity-enrichment

Research

JoVE Journal

Biology

22.7K Aufrufe.  Fred Hutchinson Cancer Research Center - FHCRC. Stabile Isotope Standards und Capture von Anti-Peptid-Antikörper (SISCAPA) Paare Affinitätsanreicherung von Peptiden mit Isotopenverdünnungsanalyse Massenspektrometrie (MRM-MS) zur quantitativen Messung von Peptiden als Ersatz für ihre jeweiligen Proteine ​​liefern. Hier beschreiben wir das Protokoll mit magnetischen Partikeln in einer teilautomatisierten Format.

Serie: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

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

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Eine quantitative Bewertung der Hefe Lipidome mit Elektrospray-Ionisation-Massenspektrometrie

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

In vivo Quantifizierung der G Protein-gekoppelten Rezeptor-Wechselwirkungen mit spektral aufgelöste Zwei-Photonen-Mikroskopie

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling von Methyltransferasen und andere S-Adenosyl- L-Homocystein-bindende Proteine ​​von Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture Compound Mass Spectrometry - Ein leistungsfähiges Tool zum Identifizieren Novel c-di-GMP Effektorproteinen

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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 ...