Siamo grati a Alain Tessier per preziosi consigli, discussioni e supporto tecnico. Riconosciamo il Centro per le applicazioni biologiche di Spettrometria di Massa presso la Concordia University di servizi eccezionali. Questo lavoro è stato sostenuto dalle concessioni dal CIHR e la NSERC del Canada. VIT è un investigatore CIHR New Research Chair e Concordia University di Genomica, Biologia Cellulare e invecchiamento.

Materiali e metodi <ol><li> Ceppi di lievito e le condizioni di crescita Il ceppo selvatico BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) era cresciuto in media ricca YEPD (1 estratto di lievito%, 2% peptone, 2% di glucosio). Le cellule sono state coltivate a 30 ° C con agitazione di rotazione a 200 rpm in beute con un rapporto &quot;fiasco volume / medio volume&quot; di 5:1. </li><li> Stoccaggio di cellule di lievito per l&#39;estrazione dei lipidi <ol><li> Una cultura da 50 ml di cellule è stato raccolto per centrifugazione a 3000 xg per 5 minuti. </li><li> Lavato due volte con acqua fredda. <ol style=" list-style-type:lower-alpha;"><li> Risospendere il pellet in 25 ml di acqua ghiacciata fredda </li><li> Cellule pellet in centrifugare a 3000 xg per 5 minuti </li></ol></li><li> Risospendere il pellet e trasferimento a tubo eppendorf </li><li> Pellet per centrifugazione, 16.000 xg per 2 minuti, rimuovere il surnatante e congelare a -80 ° C fino al momento dell&#39;uso. (Usiamo un bicchiere di alcool isopropilico conservati nel congelatore -80, azoto liquido può essere utilizzato anche) </li></ol></li><li> Reagenti Biotech cloroformio grado e metanolo sono da Sigma-Aldrich. Acidi grassi liberi e trigliceridi sono stati acquistati dalla Larodan (Malmö, Svezia). Varie specie di fosfolipidi - tra cui fosfatidilcolina, fosfatidiletanolamina, fosfatidilinositolo, fosfatidilserina, acido fosfatidico e cardiolipins - sono stati ottenuti da lipidi Avanti Polar (Alabastro, AL, USA). Ad alta velocità centrifuga tubi di vetro con tappo rivestito in teflon sono stati da Fisher. </li></ol> Estrazione dei lipidi Si tratta di una modifica del protocollo descritto da Bligh e Dyer 8. Tutte le manipolazioni con i lipidi estratto deve essere fatto utilizzando siringhe o pipette di vetro, plastica crea un segnale di grande fondo se entrano in contatto con il cloroformio. I volumi esatte non sono critiche fino a quando i rapporti di 1:2:0.8 per il cloroformio - MeOH - H 2 O al punto 3 e 2:2:1.8 per il cloroformio - MeOH - H 2 O al punto 7 sono conservati. <ol><li> Risospendere le cellule congelate in 1,6 ml di ghiacciata distillata H 2 O. </li><li> Trasferire 1,6 ml di sospensione cellulare di tubi di vetro ad alta velocità centrifuga. </li><li> Aggiungere 6 ml di cloroformio e MeOH (1:2) e miscelare 0,8 ml di perline di vetro alla sospensione cellulare. </li><li> Vortex la sospensione cellulare con perle di vetro due volte per 1 minuto. </li><li> Aggiungere 2 ml di cloroformio e mescolare delicatamente. </li><li> Incubare per 5 minuti a temperatura ambiente con miscelazione occasionali. </li><li> Aggiungere 2 ml di H 2 O distillata e mescolare delicatamente. </li><li> Incubare per 5 minuti a temperatura ambiente con miscelazione occasionali. </li><li> Centrifugare per 5 min a 3000 xg a temperatura ambiente. </li><li> Raccogliere tutta la fase liquida in una nuova ad alta velocità, tubo di vetro per centrifuga. </li><li> Aggiungere 3,2 ml di cloroformio al pellet cellulari dal punto 9. </li><li> Vortex due volte per 1 minuto. </li><li> Centrifugare per 5 min a 3000 xg a temperatura ambiente. </li><li> Aggiungere il sopranatante in fase liquida a partire dal punto 10. </li><li> Centrifugare per 5 min a 3000 xg a temperatura ambiente. </li><li> Eliminare il superiore (acquosa) di fase e il trasferimento inferiore (organici) in una nuova fase alta velocità tubo di vetro per centrifuga. </li><li> Centrifugare per 5 min a 3000 xg a temperatura ambiente. </li><li> Trasferire il surnatante in un intero flacone di vetro e secco sotto azoto. </li><li> Sciogliere il film lipidico in 500 ml di cloroformio e conservare a -20 ° C. </li></ol> Analisi dei lipidi mediante spettrometria di massa Un mix stock di standard di lipidi in cloroformio deve essere preparato in anticipo come da tabella 1, così come una soluzione stock di MeOH - cloroformio (1:1) con il 0,1% (v / v) di idrossido di ammonio. <ol><li> Prima dell&#39;iniezione, unire 10 ml di un campione con 10 microlitri della miscela standard di lipidi riportate nella Tabella 1. In 200μL di 1:1 cloroformio: metanolo con 0,1% di NH 4 OH. <ul><li> Lo standard di rapporto di campione possono essere modificati se necessario. </li></ul></li><li> Risolvere i lipidi con un Q-TOF Micromass 2 spettrometro di massa dotata di un nano-elettrospray operativi di fonte ad un flusso di 1 ml / min. <ul><li> Parametri dello strumento esatta varia da strumento a strumento. Vedere la Tabella 2 per le impostazioni di un Micromass Q-ToF 2 (Waters, Milford, MA, USA) dotata di un nano-elettrospray fonte. Anche se abbiamo sfruttato l&#39;alta risoluzione di un Q-TOF tipo di spettrometro di massa, non è un requisito obbligatorio. </li></ul></li><li> Dopo l&#39;acquisizione degli spettri di massa vengono smussate, sfondo sottratto e centrato, e quindi l&#39;elenco picco esportati in Excel. I picchi di ogni classe dei lipidi vengono poi normalizzate ai loro standard interno. A seconda dell&#39;applicazione, può essere necessario fare eseguire ulteriori post-elaborazione, come deisotoping e deconvoluzione. </li></ol> Lipidici standard Tabella 1. Interno, la loro concentrazione inil mix standard, e la modalità MS per la loro analisi. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Lipidi classe </td><td> Catena di composizione standard </td><td> Massa di standard </td><td> Concentrazione (mg / ml) </td><td> MS modalità </td></tr><tr><td> L&#39;acido fosfatidico </td><td> 14:0 / 14:0 </td><td> 591,40 </td><td> 100 </td><td> Negativo </td></tr><tr><td> Fosfatidiletanolamina </td><td> 14:0 / 14:0 </td><td> 634,45 </td><td> 200 </td><td> Negativo </td></tr><tr><td> Fosfatidilinositolo </td><td> N / A </td><td> N / A </td><td> N / A </td><td> Negativo </td></tr><tr><td> Fosfatidilserina </td><td> 14:0 / 14:0 </td><td> 622,37 </td><td> 40 </td><td> Negativo </td></tr><tr><td> Cardiolipina </td><td> 4x14: 0 </td><td> 619,92 </td><td> 100 </td><td> Negativo </td></tr><tr><td> Acidi grassi liberi </td><td> 19:00 </td><td> 297,28 </td><td> 100 </td><td> Negativo </td></tr><tr><td> Fosfatidilcolina </td><td> 14:0 / 14:0 </td><td> 650,48 </td><td> 100 </td><td> Positivo </td></tr><tr><td> Triacilgliceroli </td><td> 13:0 / 13:0 / 13:0 </td><td> 698,63 </td><td> 200 </td><td> Positivo </td></tr></tbody></table> Tabella 2. Impostazioni dello strumento per una Micromass Q-ToF 2 (Waters, Milford, MA, USA) dotata di un nano-elettrospray fonte. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> Portata </td><td> Cono di tensione </td><td> Tensione capillare </td><td> Collisione di gas </td></tr><tr><td> Modalità positiva </td><td> 1μl/min </td><td> -28 V </td><td> 3,0 kV </td><td> 10 </td></tr><tr><td> Modalità negativa </td><td> 1μl/min </td><td> 30 V </td><td> -3,2 Kv </td><td> 10 </td></tr></tbody></table>

<ol>
	<li>Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+lipokine,+a+lipid+hormone+linking+adipose+tissue+to+systemic+metabolism.">Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.</a> Cell. 134, 933-944 (2008).</li><li>Claypool, S. M., Oktay, Y., Boontheung, P., Loo, J. A., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+defines+the+interactome+of+the+major+ADP/ATP+carrier+protein+of+the+mitochondrial+inner+membrane.">Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane.</a> J. Cell Biol. 182, 937-950 (2008).</li><li>Guo, T., Gregg, C., Boukh-Viner, T., Kyryakov, P., Goldberg, A., Bourque, S., Banu, F., Haile, S., Milijevic, S., San, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+signal+from+inside+the+peroxisome+initiates+its+division+by+promoting+the+remodeling+of+the+peroxisomal+membrane.">A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane.</a> J. Cell Biol. 177, 289-303 (2007).</li><li>Russell, S. J., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+regulation+of+ageing.">Endocrine regulation of ageing.</a> Nat. Rev. Mol. Cell Biol. 8, 681-691 (2007).</li><li>Czabany, T., Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+storage+and+degradation+of+neutral+lipids+in+yeast.">Synthesis, storage and degradation of neutral lipids in yeast.</a> Biochim. Biophys. Acta. 1771, 299-309 (2007).</li><li>Br&amp;uuml, g. g. e. r., Erben, B., Sandhoff, G., Wieland, R., &amp;amp, F. T., Lehmann, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+biological+membrane+lipids+at+the+low+picomole+level+by+nano-electrospray+ionization+tandem+mass+spectroscopy.">Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectroscopy.</a> Proc. Natl. Acad. Sci. USA. 94, 2339-2344 (1997).</li><li>Schneiter, R., Br&#252;gger, B., Sandhoff, R., Zellnig, G., Leber, A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+(ESI-MS/MS)+analysis+of+the+lipid+molecular+species+composition+of+yeast+subcellular+membranes+reveals+acyl+chain-based+sorting/remodeling+of+distinct+molecular+species+en+route+to+the+plasma+membrane.">Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.</a> J. Cell Biol. 146, 741-754 (1999).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li></ol>

Questo metodo consente una rapida valutazione quantitativa del lipidome lievito utilizzando prontamente disponibili, materiali poco costosi. Il metodo permette l&#39;identificazione della maggior parte delle specie di lipidi si trovano in cellule di lievito con l&#39;eccezione di diacilgliceroli, ergosteroli ergosteryl ed esteri, anche se questi lipidi possono essere identificati sostituendo idrossido di ammonio e di idrossido di litio. Il metodo descritto permette l&#39;identificazione e quantificazione dei lipidi alle...

a quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry

I lipidi sono una delle principali classi di biomolecole e giocare ruoli importanti membrana dinamica, immagazzinamento di energia, e la segnalazione<sup&gt; 1-4</sup&gt;. Il lievito in erba<em&gt; Saccharomyces cerevisiae</em&gt;, Un organismo geneticamente e biochimicamente eucarioti unicellulari manipolabili con genoma annotati e lipidome molto semplice, è un modello valido per studiare le funzioni biologiche di specie diverse di lipidi negli eucarioti pluricellulari<sup&gt; 2,3,5</sup&gt;.<em&gt; S. Saccharomyces</em&gt; Ha 10 principali classi di lipidi di lunghezza catena principalmente di 16 o 18 atomi di carbonio e uno o nessun grado di insaturazione<sup&gt; 6,7</sup&gt;. Gli attuali metodi per l&#39;identificazione e quantificazione dei lipidi - come la cromatografia liquida ad alte prestazioni, cromatografia su strato sottile, microscopia a fluorescenza, e gas-cromatografia seguita da MS - sono ben stabiliti, ma hanno una bassa sensibilità, le varie forme sufficientemente separati molecolari dei lipidi, richiedono derivitization lipidi prima di analisi, o può essere abbastanza tempo. Presentiamo qui una descrizione dettagliata del nostro approccio sperimentale per risolvere queste limitazioni intrinseche tramite sondaggio-scan ESI / MS per l&#39;identificazione e la quantificazione del complemento di tutto di lipidi nelle cellule di lievito. Il metodo descritto non richiede la separazione cromatografica di miscele di lipidi complessi recuperato da cellule di lievito, quindi notevolmente accelerare il processo di acquisizione dei dati. Questo metodo consente l&#39;identificazione e quantificazione dei lipidi alle concentrazioni più basso g / ml ed è stato applicato con successo per valutare lipidomes di cellule di lievito e tutto il loro organelli purificato. Estrazione di lipidi da cellule di lievito tutto per l&#39;utilizzo di questo metodo di analisi dei lipidi richiede da due a tre ore. Ci vogliono solo 5-10 minuti per eseguire ogni campione di lipidi estratti e secca su un Q-TOF spettrometro di massa dotata di un nano-elettrospray fonte.

Watch this Scientific Journal Video about A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry at JoVE.com

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

Descriviamo un nuovo metodo quantitativo per l&#39;identificazione lipidomica numerose specie lipidico nel lievito con sondaggio-scan ionizzazione elettrospray spettrometria di massa (ESI / MS). Questo metodo supera attualmente i metodi disponibili per l&#39;identificazione e la quantificazione dei lipidi nella capacità di risolvere le varie forme molecolari dei lipidi, la sensibilità e la velocità.

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 ...

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Research

JoVE Journal

Biology

11.6K Views.  Concordia University. Descriviamo un nuovo metodo quantitativo per l'identificazione lipidomica numerose specie lipidico nel lievito con sondaggio-scan ionizzazione elettrospray spettrometria di massa (ESI / MS). Questo metodo supera attualmente i metodi disponibili per l'identificazione e la quantificazione dei lipidi nella capacità di risolvere le varie forme molecolari dei lipidi, la sensibilità e la velocità.

Video: A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization 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

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.

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 ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativa vivo cellulare-microscopio a fluorescenza Analisi di lievito Fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Ammide Idrogeno / Deuterio Scambio &amp; massa MALDI-TOF analisi di spettrometria di attivazione Pak2

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

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 ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Un protocollo per l&#39;identificazione di interazioni proteina-proteina Sulla base di<sup&gt; 15</sup&gt; N marcatura metabolica, immunoprecipitazione, Spettrometria di massa quantitativa e di affinità di modulazione

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Un nuovo approccio per l&#39;analisi comparativa dei multiproteico Complessi Basato su<sup&gt; 15</sup&gt; N metabolica Etichettatura e quantitativa Spettrometria di Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

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 ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Uso di dispositivi microfluidici misurare la durata della vita e cellulare fenotipi in singole cellule di lievito in erba

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Preparazione del campione per l'analisi di proteomica massa-spettrometria-basato di microvasi oculari

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...