Name: profiling thiol redox proteome using isotope tagging mass spectrometry
Uploaded: 2012-03-24T14:00:00
Duration: 12 min 7 s
Description: Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

Författarna vill tacka Dr Greg Martin (Cornell University) och hans grupp för att tillhandahålla DC3000 stammen, frön tomat och råd. De skulle också vilja tacka Dr Zhonglin Mou för hjälp med DAB protokollet och Proteomics Division på UF tvärvetenskapligt centrum för bioteknik Forskning för stöd i metodutveckling. Protokollet för proteinextraktion ändrades från Hurkman och Tanaka 16. Protokollet om cysTMT märkning, steg 4 till 6 har anpassats baserat på den ursprungliga Thermo Pierce Fisher Scientific produkthandboken 17. Detta arbete har finansierats av National Science Foundation (MCB 0.818.051 till S Chen).

1. Växande plantor och preparera bakterier <ol><li> Groddplantor groddes i MetroMix 500 jordblandning (BTI Företag) i en tillväxtkammare (160 ^ mol fotoner m -2 ar -1 med en fotoperiod av 8 timmars ljus vid 22 ° C och 16 timmars mörker vid 20 ° C under 1 vecka. Relativ fuktighet var inställd på 70%. Plantor vattnades med kranvatten efter behov för att hålla jorden från att bli torr. </li><li> Två småplantor av liknande storlek transplanteras sedan till 4 &quot;diameter krukor innehållande MetroMix 500 jord och odlades under ytterligare 3 veckor under samma betingelser som 1,1. </li><li> Pseudomonas syringae (Pst) är initialt t-ströks på Kings B Medium (KBM) plattor (per liter: 20 g proteospepton, 0,1975 g MgSO 4, 1% glycerol, 1,5 g K 2 HPO 4, 18 g agar) och odlades under två dagar vid 28 ° C. En enda koloni uppsamlas, blandas in i 300 ^ vätska Kings B-medium (20 g proteospepton, 0,1975 g MgSO 4 </sub&gt;, 10 ml glycerol, 10 ml 100x K 2 HPO 4 lager (1,5 g K 2 HPO 4 i 10 ml H 2 O), och spreds på en Kings B Medium plattan. Detta odlades över natten vid 28 ° C. </li><li> Bakterien ympämnet framställes genom att skrapa de odlade bakterierna från KBM plattan i 20 ml H2O En OD 600 av 0,03 (~ 10 6 cfu / ml) framställs i 1 L av ympning buffert (10 mM MgCl2 + 400 | il Silwett-70). Inokuleringsbuffert minus bakterier iordningställda för att reglera lösningen. </li></ol> 2. Ympning av tomat med Pst och H 2 O 2 Histokemisk Stain <ol><li> Fyra veckor gamla plantor inokulerades genom doppning i den ovan inokulering lösningen under 30 sekunder. Tydliga plastpåsar sattes över växterna omedelbart efter ympningen. </li><li> 3&#39;-3 &#39;diaminobensidin (DAB) fläck 11 framställdes (1 mg / ml DAB i H2O, pH 3,8 (HCl)) tre diays använda innan för att uppnå fullständig upplösning. Lösningen skakades för att blanda vid rumstemperatur. </li><li> Tjugofyra timmar efter behandlingen var helt expanderade bladet bort, placeras i DAB bets epidermal uppåt, och vakuum infiltrerat i 15 min. Bladen placerades därefter i mörker vid rumstemperatur över natten för färgning. </li><li> Bladen kokades i 95% etanol under 10 min, och hölls sedan i 75% etanol. </li></ol> 3. Proteinextraktion <ol><li> Efter skörd togs tomatblad maldes till fint pulver med flytande kväve med användning av mortel och mortelstöt. </li><li> För 0,5 g vävnad, tillsätt 1,25 ml Tris, pH 8,8 buffrad fenol och 1,25 ml / 0,5 g extraktionsbuffert (0,1 M Tris-HCl, pH 8,8, 10 mM EDTA, 0,9 M sackaros) och därefter fortsätta målning under ytterligare några minuter i en dragskåp. </li><li> Överför extraktet till Oakridge centrifugrör (Thermo Fisher Scientific Nalgene Company) och agitera för 2 timmar vid rumstemperatur. </li><li> Centrifugera vid 5000 xgoch 15 ° C under 10 minuter. Överföra den översta fenol fasen till ett nytt rent rör. </li><li> Back-extrahera vattenfasen med lika stor volym fenol extraherade buffert; skaka på en skakanordning under 30 min. Centrifugera och överför extraktet till en ny Falconrör. </li><li> Upprepa ovanstående steg en gång och överföra extraktet till ny Oakridge rör. </li><li> Utfälla fenol extraherade proteinerna genom tillsats av 5 volymer 0,1 M ammoniumacetat i 100% metanol (lagrad vid -20 ° C). Vortexa och inkubera vid -20 ° C under natten. </li><li> Uppsamling av proteinet pelleten genom centrifugering vid 20.000 xg, 4 ° C under 20 minuter. </li><li> Tvätta pelleten 2 ggr med kall 0,1 M ammoniumacetat i metanol och 2 gånger med 80% kall aceton. Tillsätt 1,5 ml kall 70% etanol till pellets och suspendera. Överföring till en 2 ml mikrocentrifugrör (USA Scientific) och centrifugera vid 14000 rpm, 4 ° C under 20 minuter. Avlägsna etanol. </li><li> Torka pelleten i korthet i en SpeedVac-koncentrator och upplösas i ett protein Extrhandling buffert, t.ex. ReadyPrep Protein Extraction Kit Reagens 3 (8 M urea, 4 viktprocent CHAPS, 40 mM Tris-bas, 2 M tiokarbamid) (Bio-Rad). Centrifugera vid 14000 rpm och 20 ° C under 30 min för att bilda en pellet. Samla upp supernatanten. </li><li> Mäta proteinkoncentration med användning av en CB-X-protein-analysen enligt tillverkarens manual (Geno Technology). </li></ol> 4. Provberedning och peptid märkning med cysTMTs <ol><li> Framställ proteinprov i en koncentration av 2-5 pg / pl. Här används 100 | ig protein för varje massa-tagg, 20 | il-50 jil prover. För detta experiment alla 6 hade använts. </li><li> Tillsätt lika stor volym av alkylering buffert (100 mM Tris-HCl pH 7,5, 200 mM jodacetamid) till proteinprovet att blockera den fria tiolgruppen. Bufferten bör beredas på nytt. Alkyleringen utförs vid 37 ° C under 1 timme. </li><li> Fälla ut proteinet genom tillsats av 1 ml kall 80% aceton vid -20 ° C under natten. Pelleten proteinet genom centrifugering vid 14.000rpm, 4 ° C under 20 minuter. Tvätta pellet 3 gånger med 80% aceton virvling varje gång. Avlägsna supernatanten och fick lufttorka under på is. </li><li> Återsuspendera pelleten i 50 pl lysbuffert (6 M urea, 50 mM Tris-bas, 1 mM EDTA). 0,5 ^ il av 100 mM Tris (2-karboxietyl) fosfin (TCEP) tillsätts sedan för att reducera disulfidbindningarna genom att inkubera under en timme vid rumstemperatur. </li><li> Framställa taggar genom tillsats av 20 | il av acetonitril till reagensrören att solubilisera de taggar. Vortexa och centrifugera ner till botten. </li><li> Tvätta ut extra TCEP med användning av en Microcon 3KD centrifugalfilter anordning (Millipore). Tillsätt 50 pl prov till Microcon 3KD kolonn och 50 | il lysbuffert till kolonnen. Centrifugera i 15 min vid 10.000 xg och 4 ° C. Upprepa detta steg för totalt tre gånger. Ta bort kolumn och invertera i ett rent rör. Centrifugera under 5 min vid 1000 x g och 4 ° C. </li><li> PH kontrolleras. Vid behov, justera pH till 7,0-8,0 med 1 M HCl. </li><li> Tillsätt 5 | il av den cysTMT reagens för varje prov (The cysTMT reagenskit ger 20 pl taggar för upp till 500 g protein. Denna metod använder 100 g protein, därför använder vi en femtedel av taggen.). Låta reaktionen fortskrida under 2 h vid rumstemperatur. </li><li> Prover kombinerades med Laemmli provbuffert (Bio-Rad) i en 1:1-förhållande. </li></ol> 5. Borttagning av icke reagerade-Tag Sample Fractionation <ol><li> Proverna kokades under 5 min och separerades på en förtillverkad 12% polyakrylamidgel (Bio-Rad). Gelén sköljdes tre gånger i 5 minuters intervaller med filtrerades H2O och färgades med Coomassie Blue (Bio-Rad) under 1 timme. Gelén var de-färgades över natten med H 2 O </li><li> Tolv fraktioner samlades från varje gel bana. Fraktionerna bestämdes med koncentrationer proteinband. Fraktionerna hackas till 1 mm bitar och uppsamlas i en 1,5 ml-rör. </li><li> Gelstycken var de-färgades med användning av 50% acetonitril och 0,1 M ammoniumbikarbonat i H 2 </sub&gt; O. Detta steg måste upprepas tills blåa färgen tas bort från gelbitar (cirka 3-4 gånger, 15 min varje gång). </li><li> De-färgade gelén stycken torkades med användning av 100% acetonitril (täcka gelstycken) under 15 min, supernatanten avlägsnades, och bitarna torkas med användning av en Speedvac. </li><li> Lös trypsin (Promega) vid 1:10 (trypsin: protein-förhållande) i samma volym (såsom poolade märkt prov) av 25 mM ammoniumbikarbonat. Till exempel behöver 600 | ig protein provet med en volym av 500 | il 60 ^ g trypsin upplöst i 500 | il av 25 mM ammoniumbikarbonat. </li><li> Tillsätt trypsin lösningen på gel bitar och inställd på is för att rehydrera. Om bitar inte täcks tillsätt 50 mM buffert ammoniumbikarbonat. Efter rehydrering, inkubera vid 37 ° C under 12-16 timmar. </li><li> Avlägsna vätskan proteinet utdrag ur inkuberade proverna. </li><li> Stoppa reaktionen och avlägsna eventuellt kvarvarande nedbrutet protein genom att täcka gelstycken med 5% myrsyra, 50% acetonitril .. Skaka gelstycken under 20 min vid rums temperature. Överför supernatanten till de extraherade rören proteinprov från 5,7. Upprepa extraktionen två gånger. Torka de extraherade peptider med användning av SpeedVac. </li></ol> 6. Provet Anrikning av cysTMT-märkta peptider <ol><li> Tillsätt en gradient av anti-TMT-harts till 0,5 ml rör för en 50% uppslamning. (Gradient bestämdes från bandet koncentration i fraktionering gelén. Om en fraktionsuppsamling består av en mörkt band, mer harts behövs. Fraktioner med svagare band kräver mindre harts som det finns mindre protein.) </li><li> Hartset tvättades sedan tre gånger med en kolonnvolym av 1 x Tris-buffrad saltlösning (TBS) (25 mM Tris, 0,15 M NaCl, pH 7,2) (Thermo Scientific Pierce Protein Research Products). </li><li> Tillsätt 200 pl 1xTBS till varje prov. Proverna sattes därefter till anti-TMT-harts (Thermo Scientific Pierce Protein Research Products) och omrördes vid rumstemperatur under 2 h följt av gungning över natten vid 4 ° C. </li><li> Lägg prov kolumn (TheRMO vetenskapliga Pierce Protein Research Products). </li><li> Tvätta varje kolumn tre gånger med 1 x 200 | il TBS. Detta följs sedan med tvättning tre gånger med 0,05% CHAPS (upplöst i 1 x TBS). </li><li> Kolonnen tvättas sedan tre gånger med 4 M urea i 1x TBS. Tvåhundra mikroliter av H 2 O används sedan för att tvätta kolonnen tre gånger. </li><li> Varje prov eluerades tre gånger med 200 | il 50% elueringsbuffert (50% acetonitril, 0,4% TFA). </li><li> Proverna torkades sedan i en vakuumkoncentrator. </li></ol> 7. Masspektrometrianalys <ol><li> Återsuspendera proverna i 12 | il 3% acetonitril med 0,1% myrsyra och injicera 5 pl direkt på en Eksigent NanoLC-1D högtrycksvätskekromatografi kolonn (AB SCIEX, USA). </li><li> Peptider kommer att separeras på en Proteopep II C18-kolonn 75 fim ID x 20 cm nya mål, USA) med användning av en 4-60% gradient (A: 3% acetonitril, 0,1% myrsyra; B: 97% acetonitril, 0,1% myrsyra syra)vid 3 | il / min under 60 min. </li><li> En Thermo Scientific LTQ Orbitrap XL masspektrometer användes för att detektera peptider med en topp 2 x 3 experiment som består av ett steg MS följt av förvärvet av 3 MS / MS-spektra med högre energi C-fällan dissociation (HCD) fragmentering följt av 3 MS / MS med kollision dissociation (CID) för proteinidentifiering. Parametrar i detta läge var: Isolering bredd: 3,0 m / z; Kollisionsenergin: 50% (10% i två steg). Endast dubbelt-och trippelt laddade peptider ut för fragmentering. Dynamiska utslagning parametrar in på: Upprepa count = 1; Upprepa Längd = 60, uteslutande listan size = 500; uteslutningens varaktighet = 28. Målvärden är som följer: MS = 5 E 5, MS / MS (HCD) = 1 e 5. Jonöverföring tid var satt till 500 för FTMS och 300 för MS / MS (HCD). Två microscans krävdes för HCD spektra. </li></ol> 8. Databassökning och kvantifiering <ol><li> De förvärvade CID och HCD data var ettnalyzed med Thermo Scientific Proteome Discoverer mjukvara 1,2 (Thermo Scientific Pierce Protein Research Products) genom en förgrenad arbetsflöde som genomförde en reporter Ion Quantizer (20 ppm massa tolerans fragmentjoner) att kvantifiera förhållandet. En separat segment bearbetas MS 2 spektra genom Spectrum Selector, Spectrum Normalizer och Spectrum noder Grouper. </li><li> Data har sedan sökte mot SEQUEST sökmotorn. En 20 ppm massan tolerans användes. Statiska ändringar sattes till cysTMT reagenset (304,18 Da) och dynamiska förändringar inkluderade fosforylering och metionin oxidation (15,99 Da). En anpassad databas för Solanum lycopersicum utgjordes med hjälp av RNA-data (med ca 350.000 poster från Harvard University) 13 användes i båda fallen. </li></ol> 9. Representativa resultat En representativ bild av en kontroll tomatplanta blad och en Pseudomonas inokulerat blad ärvisas i figur 1. En skillnad mellan behandlad kontroll och Pseudomonas behandlade blad observeras. Efter att bladen avlägsnas och färgades med DAB, de-färgning process möjliggör den histokemisk färgning för att visa tecken på ROS i bladvävnaden (figur 2). Figur 2A är representativ för en kontroll blad utan någon färgning. Figur 2B är representativ av ett blad som behandlats med Pseudomonas och positiv färgning för H 2 O 2-produktion. Ett exempel på Proteome Discover datautgången hos en regleras differentiellt redox-proteinet visas i Figur 3. Detta protein är en känd redox reglerad proteinet ferredoxin-en 14 och har visats spela en roll i försvaret mot Pseudomonas syringae pv tomat 15. Toppintensitet mellan kontroll och inokulerade prover användes för att erhålla relativ kvantifiering, som uppvisade signifikanta förändringar i ferredoxi-1 redox förordning (p &lt;0,05). Högintensiva toppar antyder att detta protein oxideras som svar på den patogen behandling. Figur 4 är ett exempel på Proteome Discover datautgången hos ett protein som har liknande redox reglering av ett protein mellan en kontroll och inokulerades prov. Topparna av liknande intensitet tyder på förekomst av disulfidbindningar som inte regleras av en förändring av behandlingen. Metoden kommer att revolutionera hur forskare upptäcker redox lyhörda cysteiner och disulfider 10. <img alt="Figur 1" src="/files/ftp_upload/3766/3766fig1.jpg" /> Figur 1. En representativ bild av inokulerad tomatblad med kontroll-lösning (A) och Pseudomonas (B). &lt;span class = &quot;pdflinebreak&quot;&gt; <img alt="Figur 2" src="/files/ftp_upload/3766/3766fig2.jpg" /> Figur 2. En representativ bild av DAB-färgning av inokulerad tomatblad med kontroll-lösning (A) och Pseudomonas (B). Bladen färgades med användning av 3&#39;-3 &#39;diaminobensidin. Klorofyll togs bort från blad genom kokning i 95% etanol. Mörk färgning indikerar närvaron av H2O 2. Bara lämnar ympas med bakteriekultur visade mörk färgning. <img alt="Figur 3" src="/files/ftp_upload/3766/3766fig3.jpg" /> Figur 3. Ett exempel på Proteome Discover datautgång av ferredoxin-1, differentiellt redox regleras proteinet 14. Peak intensiteten ovanför varje topp används för absolut kvantifiering. Peak intensitet mellan kontroll och inokulerade prov användsatt utföra relativ kvantifiering. <img alt="Figur 4" src="/files/ftp_upload/3766/3766fig4.jpg" /> Figur 4. Ett exempel på Proteome Discover datautgången hos ett protein som har liknande toppintensitet mellan en kontroll och inokulerades prov. Topparna av liknande intensitet tyder på förekomst av disulfidbindningar som inte regleras av en förändring av behandlingen.

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Detta protokoll innehåller information om DAB färgning samt cysTMT märkta redox cystein kvantifiering. Dessa förfaranden är till nytta för att undersöka produktion av ROS samt effekt på protein reglering när Solanum lycopersicum ympas med Pseudomonas syringae. De metoder som presenteras i detta protokoll är ett sätt att undersöka ROS i hela bladprover på ett sätt som orsakar minsta skada på bladvävnad. Märkningen Förfarandet ger ett sätt att undersöka potentiellt redox regleras proteiner genom att använda en metod cystein märkning. Detta är fördelaktigt när man undersöker ett tidigt skede av stress. Metoder som isotop-kodad affinitetsmarkör (ICAT) och cysTMT kan användas för att avgöra eventuella redox reglerade proteiner i biologiska prover. ICAT gör märkning och jämförelse av två prover 12. Båda metoderna märka fria cysteiner och kan användas för protein quantification 10,12. Emellertid tillåter cysTMT metod för en minskning i experimentell variation liksom multiplexering 10. Antalet taggar tillgängliga möjligt för forskare att ta replikerar eller flera prover i sin experimentella design. Med fler prover ger möjlighet för ett större antal identifierade proteiner. En stor nackdel med den cysTMT teknik är att den äventyrar den totala kvaliteten av proteinidentifiering grund av den selektiva anrikningen stegen för cysTMT märkta peptider (6,5-6,6). Antalet peptider för proteinidentifiering beror till stor del på antalet cysteinrester i proteinsekvensen. Detta problem kan övervinnas genom att lämna en del av den tryptiska provet före anrikning för masspektrometri proteinidentifiering. På grund av karaktären av den experimentella utformningen såväl som märkning mekanism som den cysTMT metod utnyttjar vissa steg är kritiskt. Medan du utför protein precisionpitation och pellets tvättar (3,9) är det viktigt att hålla prover kylas på is för att minska proteinnedbrytning. Under cysTMT märkning är avlägsnande av reduktionsmedel (4,6) viktig, eftersom prover kan genomgå omvänd märkning. Omvänd märkning är möjlig om reduktionsmedel kvar i provet. Om proverna reduceras efter märkning, kan cysTMT taggen avlägsnas. När väl etiketten sätts till proverna, måste pH-värdet kontrolleras (4,7) för att få optimal märkning effektivitet. Dessutom är dataanalys beror på vad som krävs av forskaren och det slutliga målet att använda protokollet. Det är också beroende av programvara som används som varje program har olika algoritmer. Detta experiment använder patogen som elicitor för ökad produktion av reaktiva oxidativa arter i tomat, men kan andra redox reglerade svar mätas i enlighet därmed. Denna experimentella designen kan anpassas till andra anläggningar och system djur. </p&gt;

<table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td></tr><tr><td> MetroMix 500 </td><td> BWI Företag </td><td> TX-500 </td></tr><tr><td> 3,3 &#39;-diaminobensidin </td><td> Sigma-Aldrich </td><td> D8001 </td></tr><tr><td> ReadyPrep Sekventiell Extraktion kit Reagens 3 </td><td> Bio-Rad </td><td> 163-2104 </td></tr><tr><td> CB-X-proteinanalysen </td><td> Geno teknik </td><td> 786-12x </td></tr><tr><td> cysTMT reagens </td><td> Thermo Scientific Pierce Protein Research Products </td><td> 90.071 </td></tr><tr><td> Laemmli provbuffert </td><td> Bio-Rad </td><td> 161-0737 </td></tr><tr><td> Bio-Safe Comassie (G-250-färg) </td><td> Bio-Rad </td><td> 161-0786 </td></tr><tr><td> Microcon 3KD kolonn </td><td> Millipore </td><td> 42.403 </td></tr><tr><td> Immobiliserade anti-TMT-harts </td><td> Thermo Scientific Pierce Protein Research Products </td><td> 90.076 </td></tr><tr><td> Centrifug kolonn </td><td> Thermo Scientific Pierce Protein Research Products </td><td> 89.896 </td></tr><tr><td> Proteopep II C18-kolonn </td><td> Nya mål </td><td> PFC7515-PP2-10 </td></tr><tr><td> NanoLC-1D HPLC </td><td> AB Sciex </td><td> 90.389 </td></tr><tr><td> LTQ-Orbitrap XL </td><td> Thermo Scientific </td><td> 0020137580 </td></tr><tr><td> SpeedVac </td><td> Labconco </td><td> 7812013 </td></tr><tr><td> Proteomanalys Discoverer 1,2 mjukvara </td><td> Thermo Scientific Pierce Protein Research Products </td><td></td></tr><tr><td> Trypsin </td><td> Promega </td><td> V5111 </td></tr><tr><td> Oakridge centrifugrör </td><td> Thermo Scientific Nalgene Företag </td><td> 3139-0050 </td></tr><tr><td> Mikrocentrifugrör (2 ml) </td><td> USA Scientific </td><td> 1620-2700 </td></tr><tr><td> 12% mini-Protean TGX Precast Gel </td><td> Bio-Rad </td><td> 456-1043 </td></tr><tr><td> Överst på Form &gt; Bio-Safe Coomassie StainBottom Form </td><td> Bio-Rad </td><td> 161-0786 </td></tr><tr><td> TMT anrikning kit </td><td> Thermo Scientific Pierce Protein Research Products </td><td> 90.077 </td></tr></tbody></table>

profiling thiol redox proteome using isotope tagging mass spectrometry

Pseudomonas syringae pv. Planta DC3000 inte bara orsakar bakteriell fläck sjukdom i Solanum lycopersicum utan även på Brassica-arter, såväl som på Arabidopsis thaliana, en genetiskt foglig värdväxt 1,2. Ackumuleringen av reaktiva syrespecies (ROS) i kotyledoner inokulerade med DC3000 indikerar en roll för ROS vid modulering nekrotisk celldöd under bakteriell sjukdom fläck av tomat 3. Väteperoxid, en komponent av ROS, framställs efter inokulering av tomatplantor med Pseudomonas 3. Väteperoxid kan detekteras med användning av en histokemisk färgning 3&#39;-3 &#39;diaminobensidin (DAB) 4. DAB-färgning reagerar med väteperoxid för att producera en brun fläck på bladvävnad 4. ROS har en reglerande roll av den cellulära redox miljön, vilket kan ändra redox status för vissa proteiner 5. Cystein är en viktig aminosyra känslig förredox förändringar. Under mild oxidation, tjänar reversibel oxidation av cystein sulfhydrylgrupper såsom redox sensorer och omformare signal som reglerar ett flertal fysiologiska processer 6,7. Tandemmasspektrometri tagg (TMT) reagens möjliggör samtidig identifiering och multiplexeras kvantifiering av proteiner i olika prov med användning av tandemmasspektrometri 8,9. De cystein-reaktiva TMT (cysTMT) reagens möjliggör selektiv märkning och relativ kvantifiering av cystein-innehållande peptiderna från upp till sex biologiska prover. Varje isobarisk cysTMT tagg har samma nominella modermassan och är sammansatt av en sulfhydryl-reaktiv grupp, en MS-neutralt spacerarm och en MS / MS-reportern 10. Efter märkning proverna föremål för proteasklyvning. De cystein-märkta peptider anrikades med användning av en harts innehållande anti-TMT-antikropp. Under MS / MS-analys, en serie reporter joner (dvs 126-131 Da) dyker upp i den låga massan regionen med information om relativ kvantifiering.Arbetsflödet är effektiv för att minska urvalet komplexiteten, förbättra dynamiskt omfång och studera cystein ändringar. Här presenterar vi redox proteomic analys av Pst behandlade DC3000 tomat (Rio Grande) lämnar hjälp cysTMT teknik. Detta högeffektiv metod har potentialen att användas för att studera andra redox-reglerade fysiologiska processer.

Watch this Scientific Journal Video about Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry at JoVE.com

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Reaktiva syrespecies-nivån är förhöjd när celler möter stressförhållanden. Här visar vi som exempel 3&#39;-3 &#39;diaminobensidin färgning liksom cysTMT märkning och masspektrometri för att profilera redox proteomanalys i<em&gt; Pseudomonas syringae</em&gt; Behandlades tomatblad.

Profilering Tiol Redox Proteome Användning Isotope spektrometri Taggning Mäss

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

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Research

JoVE Journal

Biology

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 Provberedning: Ultra tunt lager Metod

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

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.

En kvantitativ bedömning av jästen Lipidome med elektrospray Spektrometri jonisation

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.

Analysera stora proteinkomplex från struktur-masspektrometri

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

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Oligo Mass profilering (OLIMP) av Extracellulär Polysackarider

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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.

Profilering av metyltransferaser och andra S-Adenosyl- L-Homocystein-bindande proteiner av Capture Compound masspektrometri (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 ...

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.

Kvantifiering av proteiner med Peptide Immunoaffinity Anrikning kombination med masspektrometri

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

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.

Identifiering av proteinkomplex i<em&gt; Escherichia coli</em&gt; Hjälp Sekventiell rening peptid affinitet i kombination med tandem masspektrometri

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

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.

Vald Reaction Monitoring masspektrometri för Absolute protein kvantifiering

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

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identifiering av MyoD Interactome Använda Tandem Affinity Purification Kopplad till masspektrometri

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Cellulär redoxprofilering med hög innehållsmikroskopi

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...