Name: subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)
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Description: Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universita&#776;t Go&#776;ttingen.

1. Förbered en säker arbetsplats genom att betrakta biosäkerhet Villkor <ol><li> Bekanta dig med de laboratorie- och säkerhetsföreskrifter som är relevanta för att arbeta med mikroorganismer. De flesta mänskliga patogena mikroorganismer måste hanteras på skyddsnivå 2 villkor, men några, såsom Salmonella enterica serovar Typhi kräver skyddsnivå 3. Information om nivån att hantera varje patogen kan nås på www.cdc.gov/biosafety. </li><li> Oberoende av biologiskt klassificeringen av den specifik...

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Det mest kritiska steget i upprättandet av en MSPP system är entydig genetiska bestämningen av biomarkör jon identiteter. Om det inte är möjligt att identifiera en biomarkör utan tvekan, då bör uteslutas från systemet 13. C. jejuni ssp. doylei systemet omfattar 14 olika biomarkörer joner. Dessa är 5 lägre jämfört med C. jejuni ssp jejuni MSPP systemet. 13 .Det viktigaste skillnaden mellan den detekterbara C. jejuni<...

Under det senaste decenniet har matris assisterad laserdesorptionsjonisering time-of-flight masspektrometri (MALDI-TOF MS) avancerade till vara en högt värderad standardmetod för mikrobiell släkte och artbestämning i klinisk mikrobiologi 1, 2. Artbestämning baseras på registrering av små protein fingeravtryck av intakta celler eller cellysat. Den typiska massområdet för en masspektrometer som används i rutinmässig klinisk mikrobiologi är 2-20 kDa. Dessutom kan den resulterande spektra användas för att diskriminera stammar på nedanstående arter och under-underarter nivå tre. Tidiga banbrytande studier har identifierat särskilda biomarkörer joner för en särskild undergrupp av stammar i Campylobacter jejuni 4, Clostridium difficile 5, Salmonella enterica ssp. enterica serovar Typhi 6, Staphylococcus aureus 7-9 och Escherichia coli 10-12. Kombinationen av flera variabla biomarkörer massa som motsvarar alleliska isoformer ger möjlighet för djupare subtyp. Tidigare framgångsrikt genomfört vi en metod för att konvertera dessa variationer i mass profiler i meningsfulla och reproducerbara phyloproteomic relationer kallas masspektrometri baserade phyloproteomics (MSPP) på en C. jejuni ssp. jejuni isolat samling 13. MSPP kan användas en masspektrometrisk ekvivalent med DNA-sekvensbaserade subtyp tekniker som multilocus-sekvensen typning (MLST). Art av campylobacter är den vanligaste orsaken till bakteriell gastroenterit i världen 14, 15. Som en konsekvens av Campylobacteriosis post-infektiös sequela, nämligen kan Guillain-Barrés syndrom, reaktiv artrit och inflammatorisk tarmsjukdom uppstår 16. De viktigaste smittkällor ärförorenade boskap kött från kyckling, kalkon, svin, nötkreatur, får och ankor, mjölk och ytvatten 15, 17. Därför reguljära studier epidemiologisk övervakning i samband med livsmedelssäkerheten är nödvändiga. MLST är den &quot;gyllene standarden&quot; i molekylär typning för Campylobacter arter 18. Eftersom Sanger-sekvensering baserade MLST metod är arbetsintensiv, tidskrävande och relativt dyra, är MLST typning begränsad till relativt små isolat kohorter. Det finns därför ett behov av billigare och snabbare subtyp metoder. Detta behov kan tillgodoses genom masspektrometriska metoder som MSPP. Detta dokument presenterar ett detaljerat protokoll för MSPP-typning med en samling av Campylobacter jejuni ssp. Doylei isolat och jämförelse av dess potential med MLST.

<table border="0" cellpadding="0" cellspacing="0" width="1348">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">acetonitrile</td>
			<td style="width:663px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">34967</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoflex III TOF/TOF 200 system</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">GT02554 G201</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">bacterial test standard BTS</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">BioTools 3.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>263564</td>
			<td>Software Package</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Bruker IVD Bakterial Test Standard</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">8290190</td>
			<td>5 tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8843</td>
			<td>ATCC 49349;IMVS 1141;NCTC 11951;strain 093</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9143</td>
			<td>Goossens Z90</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG7790</td>
			<td>ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9243</td>
			<td>Goossens N130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8871</td>
			<td>NCTC A603/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9255</td>
			<td>Goossens B538</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8870</td>
			<td>NCTC A613/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar base&#160;</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1.10455 .0500</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Compass for FlexSeries 1.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>251419</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defibrinated sheep blood&#160;</td>
			<td>Oxoid Deutschland GmbH, Wesel, Germany</td>
			<td>SR0051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">ethanol</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">02854 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HCCA matrix</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Kimwipes paper tissue</td>
			<td style="width:663px;">Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MALDI Biotyper 2.0</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>259935</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mast Cryobank vials</td>
			<td>Mast Diagnostica, Reinfeld, Germany</td>
			<td style="width:119px;">CRYO/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MSP 96 polished steel target</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>224989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Mini Kit&#160;</td>
			<td style="width:663px;">Qiagen, Hilden, Germany</td>
			<td style="width:119px;">51304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">recombinant human insulin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">I2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">T6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">water, molecular biology-grade</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)

MALDI-TOF MS erbjuder möjligheten att differentiera vissa bakterier inte bara på arter och underarter nivå men till och med under, på stamnivå. Alleliska isoformer av de detekterbara biomarkörer joner resulterar i isolatspecifika mass skift. Masspektrometri baserade phyloproteomics (MSPP) är en ny teknik som kombinerar masspektrometriska detekterbara biomarkörer massor i ett system som tillåter avdrag för phyloproteomic relationer från isolera specifika mass skift jämfört med ett genom sekvensreferensstam. De härledda aminosyrasekvenserna används sedan för att beräkna MSPP-baserade dendrogram. Här beskriver vi arbetsflödet för MSPP genom att skriva en Campylobacter jejuni ssp. Doylei isolat samling av sju stammar. Alla sju stammar var av humant ursprung och multilocus sekvens typning (MLST) visade den genetiska mångfalden. MSPP-typning resulterade i sju olika MSPP sekvenstyper, tillräckligt återspeglar deras PHYlogenetic relationer. C. jejuni ssp. doylei MSPP systemet ingår 14 olika biomarkörer joner, mestadels ribosomala proteiner i massområdet av 2-11 kDa. MSPP kan i princip anpassas till andra masspektrometriska plattformar med en utökad massområde. Därför har denna teknik potential att bli ett användbart verktyg för stamnivå mikrobiell typning.

Tidigare har vi lyckats etablera en MSPP system för C. jejuni ssp. jejuni 13. Här syftar vi att utvidga metoden till syskon underarter C. jejuni ssp. doylei. I detta specifika inställning, sju C. jejuni ssp. doylei isolat förvärvades från den belgiska samling av mikroorganismer / Laboratory of Microbiology UGent BCCM / LMG Gent, Belgien. Alla sju isolat används för våra analyser var av humant ursprung. Genomet-sekv...

Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Masspektrometri baserade phyloproteomics (MSPP) användes för att skriva en samling av Campylobacter jejuni ssp. Doylei isolat på stamnivå i jämförelse med multilocus sekvens typning (MLST).

subtypning av<em&gt; Campylobacter jejuni</em&gt; Ssp.<em&gt; doylei</em&gt; Isolat genom masspektrometri-baserade PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

Research

JoVE Journal

Genetics

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Förbättrad Polymerase Chain Reaction-restriction fragment length polymorphism Genotypning av Toxic Pufferfish genom vätskekromatografi / masspektrometri

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Förfarande för adaptiv Laboratory Evolution av mikroorganismer med hjälp av en kemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

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Detektion av Copy Number Ändringar Använda Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

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MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

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Analys av c-KIT-ligand-promotorn med användning av kromatinimmunfällning

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Metaboliska märkning och profilering av överföring RNAs använder Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Utredning av RNA-syntes med 5-Bromouridine märkning och Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Kandidat Gene tester i klinisk kohortstudier med multiplexade genotypning och masspektrometri

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

En snabb och kvantitativ metod för posttranslationella modifieringen och Variant aktiverad mappning av peptider till genomen

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Induction and Evaluation of Inbreeding Crosses Using the Ant, Vollenhovia Emeryi

Induktion och utvärdering av inavel korsar med myran, Vollenhovia Emeryi

The genetic and molecular components of the sex-determination cascade have been extensively studied in the honeybee, Apis mellifera, a hymenopteran model ...