Questo lavoro è stato finanziato dalla Clinica Tecnologia NSC proteomica Assessment Center (CPTAC) concedere (# U24 CA126476), così come una sovvenzione da parte dell&#39;industria dell&#39;intrattenimento Foundation (FEI) e la ricerca delle donne FEI Fondo cancro al Consorzio seno Discovery Cancro biomarcatori, e generosi doni dal Keck Foundation, la Fondazione Canarie, e il Paul G. Allen Family Foundation.

Procedura sperimentale: Il test richiede peptidi sintetici e di anticorpi anti-peptide. Peptidi selezionato dovrebbe essere univoco per la proteina di interesse, contengono tra 8 e 22 amminoacidi, e non hanno conosciuto modificazioni post-traduzionali. Residui di metionina sono generalmente evitate ed i peptidi contenenti dibasico aminoacidi (es. KK, KR, RR) sono indesiderabili. Per questa tecnica, è comune utilizzare peptidi isotopo stabile etichettati come standard interni, incorporando pesante (13 C e 15 N) etichettati aminoacidi al C-terminale del peptide (cioè K o R etichettati). Il protocollo che segue descrive un test sviluppato per misurare il peptide GDSLAYGLR dal osteopontina proteina del mouse, utilizzando anticorpi anti-peptide ottenuto da Epitomics Inc. (Burlingame, CA) e peptidi sintetici dal New England Peptide (Gardner, MA). Il protocollo si compone di tre fasi principali (Figura 1): 1) la digestione della tripsina della miscela proteica complessa, 2) Arricchimento dei peptidi 3) Analisi mediante spettrometria di massa. Sarà dimostrato su un campione di plasma umano a spillo con la proteina osteopontina mouse. 1. Tripsina digestione enzimatica e pulizia <ol><li> Scongelare aliquota 10 microlitri plasma pulito in ghiaccio umido. </li><li> Determinare la concentrazione totale delle proteine ​​con il saggio BCA e centrifugare il campione per rimuovere eventuali particelle solide in sospensione. </li><li> Pipettare un&#39;aliquota 10 microlitri dal suo tubo di stoccaggio a un piatto di 1000 ml Deepwell e coprire con pellicola in grado di perforare-. </li><li> Aggiungere 20 ml di 9M fresca urea / 30mm ditiotreitolo (DTT) (finale 6M concentrazione di urea / 20 mM DTT) per ogni campione. </li><li> Incubare per 30 minuti a 37 ° C. </li><li> Aggiungere 3 ml di fresca 500 Iodoacetamide mM (finale IAM 50mm) per ogni campione. </li><li> Incubare per 30 minuti al buio a temperatura ambiente. </li><li> Aggiungere 257 ml di 100 mM Tris (pH 8) (urea diluisce a ~ 0.6M). </li><li> Aggiungere 10 ml di soluzione madre tripsina (1 mg / mL; per 1:50 enzima: rapporto di substrato). </li><li> Incubare a 37 ° C durante la notte (12-16 ore). </li><li> Aggiungere 3 ml di acido formico pulito (concentrazione finale di 1%). </li><li> Aggiungi standard di isotopi stabili (standard vengono aggiunti se si esegue un test multiplex, di solito si tratta di 10 L contenente 50-100 fmol di standard peptide isotopi marcati). </li><li> Lavare la piastra cartuccia Oasis bene con 500 microlitri di 0.1% di acido formico in 80% acetonitrile, scartando la flow-through. Ripetere questa operazione 3 volte. </li><li> Equilibrare la piastra della cartuccia con l&#39;aggiunta di 500 microlitri di 0.1% di acido formico in acqua, ed eliminare la flow-through. Ripetere questa operazione 4 volte. </li><li> Carico campioni digerire alla piastra della cartuccia e regolare il vuoto in modo che il flusso è molto lento. </li><li> Lavare con 500 microlitri di 0.1% di acido formico in acqua, ed eliminare flow-through. Ripetere questa operazione 3 volte. </li><li> Peptidi eluire con l&#39;aggiunta di 2 x 500 microlitri di 0.1% di acido formico in 80% acetonitrile in 1000 ml profondi pozzetti (non scartare la flow-through). </li><li> Liofilizzare (o speedvac) l&#39;eluato a secchezza. (Liofilizzazione è il metodo preferito) </li><li> Ricostituire secca peptidi aggiungendo 50 microlitri di PBS + 0,03% CHAPS. </li></ol> 2. Peptide immunoaffinità arricchimento <ol><li> Trasferire il campione standard di Kingfisher Piastre a 96 pozzetti. </li><li> Aggiungere 1 anticorpi mg e 1,5 microlitri proteina-G perle rivestito magnetiche per bersaglio (è facoltativo crosslink l&#39;anticorpo ai talloni prima dell&#39;aggiunta). Garantire perle sono ben sospesa agitazione o vortex. </li><li> Coprire la piastra con un foglio. </li><li> Incubare per una notte (12-16 ore) con dolci cadendo per assicurare perle sono sospesi. </li><li> Centrifugare piastra a 32 xg per 5 secondi per rimuovere il liquido dalla superficie del foglio. </li><li> Togliere il coperchio di alluminio. </li><li> Lavaggio e l&#39;eluizione delle perline può essere eseguita manualmente o in modo automatico. Questa procedura descrive le operazioni automatizzate su una piattaforma Kingfisher. </li><li> Lavare le perle 2 volte con 200 ul PBS + 0,03% CHAPS (1 minuto a lavaggio). </li><li> Lavare le perle 1 volta con 200 microlitri diluizione 1:10 di PBS + 0,03% CHAPS (1 minuto). </li><li> I peptidi sono eluiti in 25 uL del 5% di acido acetico + 0,03% CHAPS. </li><li> Posizionare la piastra eluizione su un magnete e trasferire l&#39;eluato sino ad una piastra da 96 pozzetti, facendo attenzione a non trasferire le perline avanzi. </li><li> Coprire la piastra con un tappeto di tenuta. </li><li> La piastra contenente l&#39;eluato viene consegnato allo spettrometro di massa a triplo quadrapole per l&#39;analisi. </li></ol> 3. Analisi per il monitoraggio di reazione multipla - spettrometria di massa <ol><li> Transizioni per SRM / MRM analisi possono essere selezionati e ottimizzati infondendo un 1 picomole soluzione per microlitro di peptide standard nel 30% di acido formico acetonitrile/0.1% nello spettrometro di massa a 0,5 microlitri / min. Una volta a spruzzo sia stabile, raccogliere un MS / MS dello spettro. </li><li> Le transizioni sono selezionati dalla MS / MS dello spettro, individuando three gli ioni frammento abbondante in una regione dello spettro con poco rumore. Per moltiplicare carica ioni peptide, la y-ion frammenti rilevati a m / z&gt; precursore m / z sono di solito i migliori per SRM / MRM sviluppo di analisi. Figura 2 mostra la MS / MS dello spettro e ioni di transizione selezionata 476,3&gt; 508,3, 579,3 , 692,4 (transizioni per il pesante isotopo stabile non standard di 481,3&gt; 518,3, 589,3, 702,4 indicato) per il GDSLAYGLR peptide. </li><li> A seconda della marca e del modello dello spettrometro di massa utilizzato, ci sono più parametri che possono essere ottimizzate per ogni transizione. Qui, abbiamo ottimizzato l&#39;energia di collisione di ogni transizione selezionata dalla rampa l&#39;energia di collisione e il monitoraggio del livello di segnale. Una energia di collisione di 25 è stato utilizzato per ogni transizione. </li><li> In breve, una configurazione tipica per SRM / MRM analisi è la seguente: Fase mobile (A) 0,1% acido formico; (B) Acetonitrile 90% / 0,1% di acido formico, 0,3 x 5 millimetri colonna trappola C18, 75 ID micron x 15 cm C18 colonna analitica (Reprosil-Pur C18 AQ, 120 A poro °). Il volume di iniezione è 10 L e il campione viene caricato per 5 minuti a 3% B ad un flusso totale 3 ml / min ed eluita con un gradiente lineare 3-45% B a 300 - 400 Nl / min a 10 min. Condizioni su QTRAP 4000 (ABSCIEX, Foster City, CA) sono stati spruzzo 2.3kV tensione, temperatura della sorgente di ioni a 150 ° C, GS1 di 12, e di gas tenda 15. </li><li> Il campione viene analizzato da SRM / MRM iniettando 10 ml di campione. Aree dei picchi sono integrati per peptidi leggeri e pesanti usando il programma di Skyline. 14 Calcolare il rapporto tra area del picco (PAR) del peptide senza etichetta / etichetta in ogni campione utilizzando la transizione più abbondante che è privo di rumore di fondo, in questo caso, la transizione y5 (luce peptide 476,3&gt; 579,3, pesante standard di peptide 481,3&gt; 589,3). </li></ol> 4. Rappresentante dei risultati: Misurati i rapporti delle aree dei picchi (relativamente leggero peptide endogeno di spillo peptidi marcati con isotopi pesanti) forniscono una misura quantitativa del peptide di destinazione. Figura 3 mostra un esempio di cromatogramma peptidi leggeri e pesanti in un SISCAPA arricchito campione. Si noti che i peptidi leggeri e pesanti eluire allo stesso tempo, e le transizioni multiple possono essere monitorati per ogni peptide per confermare l&#39;identità. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Figura 1"/> Figura 1. Una visione schematica del processo di SISCAPA. Una miscela proteica complessa viene digerito in peptidi. Analiti peptide mirati (analita endogeno e un isotopo stabile a spillo marcati con standard interno) sono arricchiti con anticorpi anti-peptide immobilizzato sulla proteina G particelle magnetiche rivestite. Dopo aver isolato, i peptidi mirate sono eluiti dalle particelle magnetiche e analizzati mediante spettrometria di massa per la quantificazione relativa allo standard interno. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Figura 2"/> Figura 2. MS / MS spettro della GDSLAYGLR peptide che mostra la selezione di tre frammenti di SRM / MRM transizioni. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Figura 3"/> Figura 3. Cromatogrammi Esempio di profilo di un picco analita peptide luce (rosso) e la pesante isotopo stabile marcati con standard interno (blu). Cromatogrammi per la transizione Y5 dal peptide luce (476,3&gt; 579,3) e pesante isotopo stabile etichetta standard (481,3&gt; 589,3) vengono riportati nel corso del tempo come eluire dal sistema di cromatografia.

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Nessun conflitto di interessi dichiarati.

La fase più critica nel protocollo come descritto è garantire le perle rimangono ben mescolato durante il periodo di incubazione. Permettere le perline di depositarsi sul fondo del pozzo / flacone si tradurrà in una maggiore variabilità. E &#39;anche importante spin-down qualsiasi liquido che può rimanere sulla parte superiore del pozzo / flacone dopo il periodo di incubazione. Digestione tripsina riproducibile è anche critico. La procedura per la digestione qui descritto è stato utilizzato ampiamente nel nostro laboratorio in collaborazione con SISCAPA, tuttavia, è probabile che i metodi alternativi di digestione potrebbe essere ottimizzata per un dato insieme di proteine ​​obiettivo del 15 eluizione di anticorpi liberi non interferisce con il rilevamento di. peptide, probabilmente perché l&#39;anticorpo è esclusa dalla colonna o eluisce oltre i peptidi, ma l&#39;anticorpo può essere reticolato di perline G della proteina prima di arricchimento affinità in esperimenti di grandi dimensioni o se anticorpi libero diventa un problema. Abbiamo anche ritenuto utile mettere un magnete sotto la piastra campione sul autocampionatore così come la colonna di lavaggio trappola in direzione inversa del flusso (rispetto al carico) per rimuovere eventuali residui di sfere o particelle. Oltre al metodo descritto perla magnetica, la tecnica può anche essere adattato a un formato di colonna (cioè cromatografia di affinità). 12, 16 Una volta che l&#39;utente sta bene con il protocollo generale, la tecnica è anche suscettibile di varie modifiche che migliorano il test complessivo. 11 In primo luogo, è possibile analizzare analiti multipli in un singolo test combinando anticorpi nella fase di arricchimento (cioè multiplexing). Lo spettrometro di massa è in grado di analizzare simultaneamente un gran numero di analiti. In secondo luogo, aumentare il volume del campione originale migliora la sensibilità del test.

<table border="1"><tbody><tr><th> Nome del materiale </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> 1000ul piastre pozzo profondo 96-well </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc cartuccia piatto, set-pak, 40 mg, 96 pozzetti </td><td> Acque </td><td> 186003966 </td></tr><tr><td> Kingfisher 96 pozzetti </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> Cancella 96 pozzetti, piatto bianco muro </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> Foglio di copertura per 96 pozzetti </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Axymat tappeto di tenuta per 96 pozzetti </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X perforare pellicola </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher processore perla magnetica con testa magnete PCR </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tabella 1: Materiali <table border="1"><tbody><tr><th> Nome del materiale </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> Urea </td><td> Sigma Aldrich </td><td> U6031 </td><td></td></tr><tr><td> Trizma di base </td><td> Sigma Aldrich </td><td> T1503 </td><td></td></tr><tr><td> Ditiotreitolo (DTT) </td><td> Perforare </td><td> 20291 </td><td> &quot;Senza peso ditiotreitolo&quot; </td></tr><tr><td> Iodoacetamide (IAM) </td><td> Sigma Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Tripsina Oro </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Proteina G perline magnetiche, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Tampone fosfato salino (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> L&#39;acido formico </td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td> Acetonitrile </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> Acido acetico </td><td> Sigma Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tabella 2: Reagenti <table border="1"><tbody><tr><th> Soluzione </th><th> Commenti (opzionale) </th></tr><tr><td> 100 mM Tris pH 8 </td><td></td></tr><tr><td> 9 M urea / 30 mM DTT in 100 mM Tris (pH 8) </td><td> deve essere preparata fresca ogni volta </td></tr><tr><td> IAM 500 mM a 100 mM Tris (pH 8) </td><td> deve essere preparata fresca ogni volta </td></tr><tr><td> Tripsina 1mg/mL in 100 mM Tris pH 8 </td><td></td></tr><tr><td> Tripsina inibitore 1mg/mL in 100 mM Tris pH 8 </td><td></td></tr><tr><td> Isotopo stabile di serie Mastermix </td><td></td></tr><tr><td> Anticorpi anti-peptide Mastermix </td><td></td></tr></tbody></table> Tabella 3: Soluzioni da preparare

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

C&#39;è un grande bisogno di analisi quantitative per la misurazione delle proteine. Immunodosaggi panino tradizionale, in gran parte considerato il gold standard nella quantificazione, sono associati ad un alto costo, tempi lunghi, e sono piene di inconvenienti (ad esempio gli anticorpi eterofili, le interferenze di autoanticorpi, &#39;gancio-effetto&#39;) 1. Una tecnica alternativa è l&#39;arricchimento affinità di peptidi accoppiata con spettrometria di massa quantitativa, comunemente indicata come SISCAPA (norme isotopo stabile e Capture da anticorpi anti-peptide). 2 In questa tecnica, l&#39;arricchimento affinità di peptidi con la diluizione degli isotopi stabili e il rilevamento da parte selezionato / spettrometria di massa di reazione più di monitoraggio ( SRM / MRM-MS) fornisce la misura quantitativa di peptidi come surrogati per le loro rispettive proteine. SRM / MRM-MS è ben definito per la quantificazione accurata di piccole molecole 3, 4 e più recentemente è stato adattato per misurare le concentrazioni di proteine ​​nel plasma e lisati cellulari. 5-7 Per ottenere la quantificazione delle proteine, le molecole più grandi sono digeriti a peptidi componente utilizzando un enzima come la tripsina. Uno o più peptidi selezionati la cui sequenza è unica per la proteina bersaglio in questa specie (cioè &quot;proteotypic&quot; peptidi) vengono poi arricchite dal campione utilizzando anticorpi anti-peptide e misurato come surrogati stechiometrico quantitativa per la concentrazione di proteine ​​nel campione. Quindi, insieme alla diluizione degli isotopi stabili (SID) metodi (cioè un spillo in isotopi stabili etichettati peptide standard), SRM / MRM può essere usato per misurare le concentrazioni di peptidi proteotypic come surrogati per la quantificazione delle proteine ​​in matrici biologiche complesse. I saggi hanno diversi vantaggi rispetto ai tradizionali saggi immunologici. I reagenti sono relativamente meno costosi da produrre, la specificità per l&#39;analita è eccellente, il test può essere altamente multiplex, l&#39;arricchimento può essere effettuato dal plasma pulito (senza esaurimento richiesto), e la tecnica è suscettibile di una vasta gamma di proteine ​​o modifiche di interesse. 8-13 In questo video si dimostra il protocollo di base adattata ad una piattaforma magnetica tallone.

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

Standard di isotopi stabili e cattura da anticorpi anti-peptide (SISCAPA) affinità arricchimento coppie di peptidi con diluizione degli isotopi stabili e spettrometria di massa (MRM-MS) per fornire la misura quantitativa di peptidi come surrogati per le loro rispettive proteine. Qui descriviamo il protocollo utilizzando particelle magnetiche in un formato parzialmente automatizzato.

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa

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 Views.  Fred Hutchinson Cancer Research Center - FHCRC. Standard di isotopi stabili e cattura da anticorpi anti-peptide (SISCAPA) affinità arricchimento coppie di peptidi con diluizione degli isotopi stabili e spettrometria di massa (MRM-MS) per fornire la misura quantitativa di peptidi come surrogati per le loro rispettive proteine. Qui descriviamo il protocollo utilizzando particelle magnetiche in un formato parzialmente automatizzato.

Video: 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 Preparazione del campione: il metodo ultra sottile strato

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

Ricerca

Biologia

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.

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

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.

Analizzando complessi proteici mediante spettrometria di massa di grandi dimensioni strutturali

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.

Quantificazione in vivo di G interazioni proteina recettore accoppiato con spettralmente risolte microscopia a due fotoni

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.

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (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 tiolo Proteome Redox Utilizzando tag spettrometria di massa isotopica

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.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

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 spettrometria di massa Compound - un potente strumento per identificare nuovi c-di-GMP effettori Proteine

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.

Reazione selezionato Monitoraggio spettrometria di massa per Absolute Protein Quantificazione

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.

Arricchimento della matrice extracellulare proteine ​​da tessuti e digestione in Peptidi per l&#39;analisi Spettrometria di Massa

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

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa

Quantificazione delle proteine Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di massa