Name: an aquatic microbial metaproteomics workflow: from cells to tryptic peptides suitable for tandem mass spectrometry-based analysis
Uploaded: 2015-09-15T14:00:00
Description: Watch this Scientific Journal Video about An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis at JoVE.com

The authors would like to acknowledge Marcos Di Falco for his expertise and advice with the preparation of the samples for nano-LC MS/MS as well as Dr. Zoran Minic from the University of Regina for the LC MS/MS analysis. This work was supported by NSERC (DG402214-2011) and CRC (950-221184) funding. D.C. was supported by Concordia Institute for Water, Energy, and Sustainable Systems and FQRNT.

1. Forbered Reagenser <ol><li> Forbered SDS-ekstraktionsopløsning: 0,1 M Tris-HCI, pH 7,5, 5% glycerol, 10 mM EDTA og 1% SDS. Filtersteriliser ved anvendelse af et 0,22 um filter og opbevares ved 4 ° C. </li><li> Der tilberedes nødvendige reagenser til polyacrylamidgel. <ol><li> Udarbejde 1.5 M Tris-HCI pH 8,8. Filtersteriliser ved anvendelse af et 0,22 um filter og opbevares ved stuetemperatur. </li><li> 0,5 M Tris-HCI pH 6,8. Filtersteriliser ved anvendelse af et 0,22 um filter og opbevares ved stuetemperatur. </...

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Prøve bevarelse er nøglen til metaproteomic undersøgelser og tidligere arbejde viste, at et RNA-stabilisering løsning er et nyttigt lagerbuffer til lagring af celler før proteinekstraktion 28. Ideelt set ville prøverne bevaret in situ at negere skift i proteinekspression under håndtering 33,34. Faktisk har in situ blevet udviklet prøvetagning og fiksering teknologier, som gør det muligt for den autonome indsamling og bevaring af prøver med skib-indsat instrumenter. , Adg...

Mikroorganismer er allestedsnærværende og spille vigtige roller i Jordens biogeokemiske kredsløb 1. I øjeblikket er der mange molekylære metoder til rådighed til at karakterisere mikrobielle samfunds struktur og funktion. Mest almindelige er analysen af 16S rRNA gensekvenser PCR-amplificeret fra miljømæssig DNA 2-4. En ulempe ved 16S rRNA-genet analyse er, at den kun indeholder oplysninger om fylogenetisk identitet og fællesskab struktur, med lidt information om metabolisk funktion. I modsætning hertil nærmer såsom metagenomet, metatranscriptomics og metaproteomics give oplysninger om samfunds struktur og metabolisme. Metagenomics eller analyse af indholdet af en samling af organismer gen, giver oplysninger om strukturen og funktionelle potentiale af fællesskabet 5-8. Selv kraftige, kan denne funktionelle potentiale ikke svarer til den metaboliskeaktiviteter organismerne. En organismes genotype er repræsenteret ved dets gener, som hver især kan transkriberes til RNA og videre oversat til protein, hvilket resulterer i en fænotype. Således at hjælpe med forståelsen af mikrobiel funktionel aktivitet i et miljø, bør postgenomiske analyse udføres 9. Metatranscriptomics eller analyse af RNA-transkripter er nyttig, fordi den afslører hvilke gener transskriberes i ethvert givet miljø. Men mRNA-niveauer ikke altid matcher deres tilsvarende protein niveauer på grund af translationel regulering, RNA halveringstid, og det faktum at der kan genereres flere protein kopier for hver mRNA 10. Af disse grunde metaproteomics er nu anerkendt som et vigtigt redskab for miljømæssig mikrobiologi. Fælles metaproteomic analyser bruge et haglgevær proteomisk tilgang, hvor den nærmeste fuldtallige proteiner i en kompleks prøve renses og analyseret samtidigt, som regel gennem enzymatic fordøjelse i peptider og analyser på et massespektrometer. Efterfølgende tandem massespektrometri (MS / MS) &quot;peptid fingeraftryk&quot; anvendes til at bestemme peptidsekvensen og potentielle protein af ved protein databasesøgning (for en gennemgang se 11). Proteomisk arbejde er kommet langt i de seneste 25 år takket være stigningen i genomisk datatilgængelighed og stigningen i følsomheden og nøjagtigheden af massespektrometre giver mulighed for high-throughput protein identifikation og kvantificering 11,12. Da proteiner er slutproduktet af genekspression, kan metaproteomic data til at bestemme hvilke organismer er aktive i en given miljø, og hvilke proteiner de udtrykker. Dette er en fordel, når de forsøger at bestemme, hvordan et bestemt sæt af miljømæssige variabler påvirker fænotypen af ​​en organisme eller samfund. Tidligt blev MS / MS-baserede metaproteomic studier i havet anvendes til at identificere specifikke proteiner i målrettetmikrobielle slægter, med den første undersøgelse med fokus på lys drevne protonpumpe proteorhodopsin i SAR11 marine bakterier 13. For nylig har komparative metaproteomic analyser belyst differentielle protein ekspressionsmønstre mellem komplekse samfund. Eksempler omfatter identifikation af tidsmæssige forskydninger i stofskiftet i de kystnære nordvestlige Atlanterhav 14 eller den antarktiske halvø 5. Andre undersøgelser har beskrevet variationer i protein ekspressionsmønstre på tværs rumlige skalaer, for eksempel langs en ​​geografisk transect fra en lav-næringsstof ocean gyre til en meget produktiv kyst upwelling systemet 15. For yderligere anmeldelser af metaproteomics anbefaler vi Schneider et al. (2010) 9 og Williams et al. (2014) 16. Målrettede proteomics har også været ansat i de seneste år at kvantificere ekspressionen af specifikke metaboliske veje i miljøet 17,18. There er tre hovedfaser i metaproteomic analyse. Den første fase er prøveforberedelse, som omfatter prøveindsamling, cellelyse og koncentrationen af ​​protein. Indsamling af prøver i marine mikrobiologi ofte indebærer filtrering af havvand gennem et pre-filter for at fjerne større eukaryote celler, partikler og partikel-associerede bakterier, efterfulgt af filtrering til opsamling af frie levende mikrobielle celler, almindeligvis med anvendelse af en 0,22 um patron filterenhed 19,20. Disse filtre er incased i en plastik cylinder og cellelyse og proteinekstraktion protokol, der kan udføres inden for filterenheden ville være et værdifuldt værktøj. Når biomassen stammer, skal cellerne lyseres for at muliggøre protein ekstraktion. Adskillige fremgangsmåder kan anvendes, herunder guanidin-HCI lysis 21 og natriumdodecylsulfat (SDS) -baserede lysis metoder. Selvom rengøringsmidler som SDS er meget effektive til at forstyrre membraner og solubilisering mange protein typer, Concentrations så lav som 0,1% kan forstyrre nedstrøms protein fordøjelse og MS-analyse 22. Af stor bekymring er de negative virkninger af SDS på trypsinfordøjelse effektivitet, opløsningsevne af omvendt fase væskekromatografi og ion suppression eller akkumulering inde i ionkilden 23. Den anden fase er fraktionering og analyse, hvor proteiner underkastes enzymatisk fordøjelse efterfulgt af LC MS / MS-analyse, hvilket resulterer i am / z fragmentering mønster, der kan anvendes til at fastslå den primære aminosyresekvens af den oprindelige tryptiske peptid. Forskellige fordøjelse metoder kan udføres afhængigt af de typer af rengøringsmidler samt downstream massespektrometri arbejdsgang. I vores protokol, er 1-D PAGE-elektroforese efterfulgt af fjernelse af SDS fra gelen udnyttes for at fjerne enhver forurening detergent. Analysen af ​​proteiner som er vanskelige at solubilisere, såsom membranproteiner, kræver anvendelse af høje koncentioner af SDS eller andre rengøringsmidler. Dette fører til kompatibilitetsproblemer med SDS-gelelektroforese. Hvis formålet med en undersøgelse kræver solubilisering af disse svært at solubilisere proteiner, kan rør-gel-systemet anvendes 22,24. Røret-gel-metoden omfatter proteiner i gelmatrixen uden brug af elektroforese. Efterfølgende nogen rengøringsmidler, der anvendes til opløsning fjernes før protein fordøjelse. Den tredje fase er den bioinformatiske analyse. I denne fase er søgte MS / MS peptid data mod en database oversatte nukleotidsekvenser at bestemme, hvilke peptider og proteiner er til stede i prøven. Identifikationen af ​​peptider er afhængig af databasen der søges imod. Marine metaproteomic data er almindeligt søges mod databaser består af reference- genomer, metagenomisk data såsom Global Ocean Sampling datasæt 25, såvel som enkelte celle forstærkes genomer fra udyrkede lineages 26,27. Proteinidentifikation kan også øges ved inddragelse af metagenomisk sekvenser fra den samme prøve som metaproteomic data stammer 5. Her giver vi en protokol til generering af peptider, der er egnede til MS / MS-baseret analyse fra mikrobielle biomasse indsamlet af filtrering og gemt i en RNA stabilisering løsning. Protokollen beskrevet her tillader DNA og protein, der skal isoleres fra den samme prøve, således at alle trin, der fører op til proteinet og DNA-udfældninger er identiske. Fra et praktisk perspektiv er mindre filtrering nødvendig, da kun det ene filter er påkrævet for både protein og DNA-ekstraktion. Vi vil også gerne anerkende, at denne protokol blev skabt gennem en kombination, tilpasning og ændring af to tidligere publicerede protokoller. Cellelysis trin er tilpasset fra Saito et al. (2011) 28 og i-gel trypsinnedbrydningsprodukt komponent er tilpasset fra Shevchenko et al. (2007) 29.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sterivex -GP 0.22 &#956;m filter unit</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>Bio Basic</td>
			<td>77-86-1 or TB0196-500G</td>
			<td>Protein Extraction/ SDS PAGE gel</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-1G</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio Basic</td>
			<td>15-21-3</td>
			<td>Protein Extraction/ SDS PAGE gel</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bio Basic</td>
			<td>6381-92-6</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>10K Amicon Filter</td>
			<td>Millipore</td>
			<td>UFC801024</td>
			<td>Protein Extraction</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179337-4L</td>
			<td>Protein Precipitation</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>67-64-1</td>
			<td>Protein Precipitation</td>
		</tr>
		<tr>
			<td>MPC Protein Precipitation reagent</td>
			<td>Epicenter</td>
			<td>mmP03750</td>
			<td>DNA Precipitation</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher Scientific</td>
			<td>67-63-0</td>
			<td>DNA Precipitation</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td>DNA Quantification</td>
		</tr>
		<tr>
			<td>Qubit Protein Assay kit</td>
			<td>Life Technologies</td>
			<td>Q33211</td>
			<td>Protein Quantification</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Bio Basic</td>
			<td>57-50-1</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio Rad</td>
			<td>161-0800</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>APS</td>
			<td>Bio Rad</td>
			<td>161-0700</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>30% Acrylamide</td>
			<td>Bio Rad</td>
			<td>161-0158</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio Rad</td>
			<td>161-0718</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>B-mercaptoethanol</td>
			<td>Bio Basic</td>
			<td>60-24-2</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Laemmli Sample Buffer</td>
			<td>Bio Rad</td>
			<td>161-0737</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Kaleidoscope Ladder</td>
			<td>Bio Rad</td>
			<td>161-0375EDU</td>
			<td>SDS PAGE gel</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>VWR</td>
			<td>CABDH6044-4</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>NH4HCO3</td>
			<td>Bio Basic</td>
			<td>1066-33-7</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-1G</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F0507-500ML</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>HPLC grade H2O</td>
			<td>Sigma-Aldrich</td>
			<td>270733-4L</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Bio Basic</td>
			<td>144-48-9</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5111</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 1.5ml</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>2mL ROBO vial 9mm</td>
			<td>Candian Life Science</td>
			<td>VT009/C395SB</td>
			<td>In-gel Trypsin digest</td>
		</tr>
		<tr>
			<td>PP BM insert, No spring</td>
			<td>Candian Life Science</td>
			<td>4025P-631</td>
			<td>In-gel Trypsin digest</td>
		</tr>
	</tbody>
</table>

an aquatic microbial metaproteomics workflow: from cells to tryptic peptides suitable for tandem mass spectrometry-based analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

Som en demonstration, vi udførte protokollen om to havvand prøver indsamlet fra overfladen og den maksimale af det kystnære hav i det nordlige Canada klorofyl. Mens på havet, blev 6-7 liter havvand ledes gennem en 3 um GF / D forfilter, derefter mikrobielle celler blev opsamlet på et 0,22 um patronfilter enhed ifølge protokollen af Walsh et al. 20. Celler blev straks opbevares i en RNA stabilisering opløsning, indtil yderligere bearbejdning. Efter vender tilbage til laboratoriet, vi u...

Watch this Scientific Journal Video about An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis at JoVE.com

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

En Aquatic Mikrobiel Metaproteomics Workflow: Fra celler til Tryptic Peptider Egnet til Tandem-massespektrometri-baseret analyse

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Research

JoVE Journal

Environment

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Karakterisering og anvendelse af passive samplere for Overvågning af pesticider i vand

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Alternative Method of Removing Otoliths from Sturgeon

Alternativ metode til at fjerne Otolither fra Sturgeon

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Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

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A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

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Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

Grundforskning i Plasma medicin - en overførselshastighed tilgang fra væsker til celler

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Characterization of Aquatic Biofilms with Flow Cytometry

Karakterisering af akvatiske biofilm med flowcytometri

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Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Isolering og analyse af mikrobielle samfund i jord, rhizosfære og rødder i flerårig græs eksperimenter

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

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Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Sammensætning og Distribution analyse af Bioaerosols Under forskellige miljøforhold

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Identifikation af pr- og stofferne kemisk arter med en kombineret målrettet og ikke-målrettet-Screening høj opløsning massespektrometri arbejdsproces

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...