Name: identification of fatty acids in bacillus cereus
Uploaded: 2016-12-05T15:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

Die Autoren sind dankbar, dass Thomas Mison für seine technische Unterstützung und Rachel Kopec für das Manuskript zu revidieren.

1. Bakterienkulturen <ol><li> Bereiten Sie einen Rasen der Bakterien (Bacillus cereus Stamm ATCC 14579) durch Spreizen 100 ul einer Übernachtkultur des bei 30 ° C inkubiert Stamm in LB (Luria-Bertani - Medium), über die Oberfläche einer Platte von LB - Agar - Medium. Die Platte über Nacht bei 30 ° C. </li></ol> 2. ECL: Entspricht Kettenlänge <ol><li> Berechnen ECL wie folgt: <img alt="Gleichung" src="/files/ftp_upload/54960/54960eq1.jpg" /> mit: i, der gelöste Stoff von Interesse; n die Kohlenstoffzahl der geradkettigen gesättigten Fettsäuremethylester vor solute Eluieren I; n + 1, die Kohlenstoffzahl der geradkettigen gesättigten Fettsäuremethylester nach solute Eluieren I; RTI, Rtn, Rt (n + 1) die Retentionszeiten der FAME-Spitzen oben beschrieben. HINWEIS: Erhalten der Retentionszeiten von geradkettigen gesättigten Fettsäuren Methylester durch Injektion einer Mischung von Standards (BEIN ICH). </li></ol> 3. FAME Herstellung und Analyse <ol><li> Um Lipid Fettsäuren, Ernte Bakterienzellen zu erhalten , indem Kolonien von der Agar - Platte und übertragen 40 mg (Frischgewicht, das entspricht 10 9 lebenden Zellen) von Bakterien in einem 10 ml Glasröhrchen mit Schraubverschluss und PTFE - Dichtungen Schaben. </li><li> Führen Sie die Umesterung über die Esterbindung Methode 14,15 unten detailliert beschrieben. <ol><li> 5 ml 0,2 M KOH in Methanol zu den frischen Bakterienzellen und Inkubieren bei 37 ° C für 1 Stunde. Diese Reaktion besteht aus alkalischer Methanolyse, die Esterbindung in der Lipid Brechen und Fettsäuremethylester zu erzeugen. </li><li> 1 ml 1 M Essigsäure den pH-Wert auf 7,0 zu senken. Überprüfen Sie den pH-Wert mit pH-Teststreifen. </li><li> 3 ml Hexan FAMEs zu extrahieren. </li><li> Übertragen Überstand (organische Phase) in saubere Röhrchen und konzentriert durch Abdampfen bei Raumtemperatur unter einem kontinuierlichen Stickstoffstrom etwa 200 &amp; mgr; l zu erhalten,Extrakt. Übertragen Sie die Probe in ein GC-Fläschchen mit Einsatz. </li><li> Injizieren Extrakte in eine Gaschromatographie-Massenspektrometrie (GC-MS) System. </li></ol></li></ol> 4. GC / MS-Bedingungen <ol><li> Injizieren FAME Proben in ein GC-MS-Gerät ausgestattet mit einer Kapillarsäule ZB-WAX (Länge, 30 m, Durchmesser 0,25 mm; Filmdicke, 0,25 &amp; mgr; m). </li><li> Stellen Sie die Einspritzöffnung (in splitless Modus) Temperatur auf 250 ° C. Verwenden Helium als Trägergas mit einer linearen Geschwindigkeit von 37 cm / sec. Halten der Ofentemperatur bei 50 ° C für 1 min, erhöht auf 190 ° C mit einer Geschwindigkeit von 20 ° C / min, und weitere Steigerung auf eine Endtemperatur von 230 ° C mit einer Rate von 2 ° C / min. </li><li> Für die MS, notieren Sie die Massenspektren von Elektronen-Ionisation (EI) bei 70 eV, und stellen Sie den Erwerb des gesamten Ionenstrom zwischen 50 und 400 atomaren Masseneinheiten (amu) (2 Scans / s). </li><li> Wenn es erforderlich ist, injizieren excep DMOX und Picolinyl Derivate unter den gleichen Bedingungent die Temperaturprogramm Ofen wie folgt: DMOX: 50 ° C (1 min), 20 ° C / min bis 210 ° C und 2 ° C / min bis 240 ° C (5 min); Picolinyl: 6 ° C (1 min), 20 ° C / min bis 220 ° C und 2 ° C / min bis 250 ° C (20 min). </li></ol> 5. Picolinyl Ester Herstellung von FAME 16 <ol><li> Man dampft das FAME Auszug aus Kapitel 3 mit einem Stickstoffstrom (mindestens 10 mg trockenes Material) und in 1 ml trockenem Dichlormethan aufzulösen. </li><li> Bereiten einer 1,0 M Lösung von Kalium - ter t-butylat in Tetrahydrofuran. </li><li> Fügen Sie den FAME-Extrakt und 0,2 ml 3-pyridinemethanol bis 0,1 ml Lösung hergestellt in Schritt 5.2. </li><li> Die Lösung wird bei 40 ° C für 30 min in einem geschlossenen Glasfläschchen. </li><li> Nach dem Abkühlen auf Raumtemperatur abgekühlt, gereinigt VE - Wasser (2 ml, siehe Materialien Tabelle) und Hexan (4 ml). Mischen Sie mit einem Wirbel, erlauben Phase zu trennen und sammeln die organische Phase ab. </li><li> DRy durch wasserfreiem Natriumsulfat, bis die organische Phase Zugabe ist vollkommen klar. Bringen Sie es in ein sauberes Röhrchen. Dann verdampfen zu 200 ul. Übertragen Sie die Probe in ein GC-Fläschchen mit Einsatz. </li></ol> 6. DMOX Herstellung von FAME 17 <ol><li> Man dampft das FAME Auszug aus Kapitel 3 mit einem Stickstoffstrom (mindestens 10 mg trockenes Material). </li><li> Zum Trockenextrakt FAME, fügen 250 mg 2-Amino-2-Methyl-1-Propanol. Spülen Sie das Gefäß mit Stickstoff, fügen Sie einen Stopper, und legen Sie sie in einem Heizblock über Nacht bei 190 ° C. </li><li> Nach dem Abkühlen auf Raumtemperatur wurden 3 ml Dichlormethan zu dem Rohr und 5 ml gereinigtes deionisiertes Wasser (siehe Materialien Table). </li><li> Schütteln Sie für die Phasentrennung und entfernen Sie dann die wässrige Phase. </li><li> Die organische Phase wird mit 5 ml Wasser. Schütteln Sie für die Phasentrennung und entfernen Sie dann die wässrige Phase. </li><li> Trocknen mit wasserfreiem Natriumsulfat, bis die organische Phase Zugabe ist vollkommen klar undübertragen sie in ein sauberes Röhrchen. Man dampft unter einem Stickstoffstrom, bis ein Volumen von 200 &amp; mgr; l zu erreichen. Übertragen Sie die Probe in ein GC-Fläschchen mit Einsatz. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Die FAs Chromatogrammprofile in Tabelle 1 dargestellten entsprechen B. cereus ATCC 14579 auf einer Agarplatte Oberfläche gewachsen. Ähnliche Profile wurden erhalten , wenn das Bakterium in belüfteten flüssigen Medien bei der gleichen Temperatur 8 gezüchtet wurde. Im Falle von in flüssigen Medien gezüchtet Bakterien, die bakterielle Biomasse durch Zentrifugation von dem Wachstumsmedium gesammelt und kann nach zuvor beschriebenen Protokollen gewaschen werden je nach Wachstumsbed...

Fettsäuremethylester (FAME), Gaschromatographie (GC) ist ein wesentliches Verfahren zur Lipid Charakterisierung. Sie trennt sich rasch und quantifiziert die verschiedenen Fettsäuren (FAs) einer Probe nach einer kurzen Extraktionsschritt. Derivate von Methylester sind sehr volatil, stabil und inert gegenüber der chromatographischen Säule, wodurch vermieden wird Tailing Spitzen. Ihre Identifizierung ist ziemlich einfach, wenn die Probe von bekannten FAs besteht, weil die chromatographischen Profile entweder veröffentlicht oder im Vergleich zu Standards. Darüber hinaus ist die wiederholte Injektion von Kalibrierungsstandards zur Quantifizierung verschiedener FAs nicht erforderlich, da die fast konstante Reaktion auf Flammenionisationsdetektion (FID) 1. Neben FID stellt Massenspektrometrie (MS) Detektion einer komplementären Reihe von Informationen zu FAME bestätigen. Wenn jedoch FAMEs berechnet werden unter Verwendung Elektronenionisation (EI), die resultierenden Spektren erlauben nicht immer für the Identifizierung von FA Feinstruktur. Beispielsweise Verzweigungsposition (dh eine verzweigte Methylgruppe) ist schwer vorherzusagen , da die diagnostische Ionen schwer 1 und die charakteristische Änderung Ionenabundanz in Ziel zu erfassen ist maschinenabhängig, was die Verwendung von Massenspektren Bibliotheken 2 verhindert wird . Eine weitere Herausforderung liegt in der Doppelbindung Position zu identifizieren, weil EI-Doppelbindung Migration verursacht. So FA Isomere mit Doppelbindung Positionen unterschiedlicher nicht durch ihre Massenspektren unterschieden werden. Glücklicherweise andere Werkzeuge wurden für FA Identifikation entwickelt. Zum Beispiel kann die Anwesenheit und die Position oder der Doppelbindungen in FAs Verzweigungs kann durch Berechnen der äquivalenten Kettenlänge (ECL) 3 gemutmaßt werden. Andere Derivatisierung Verfahren führen zu unterschiedlichen Massenspektren, abhängig von der Position einer Doppelbindung oder einer verzweigten Methylgruppe ist. 4,4-Dimethyl oxazolinderivaten (DMOX) 4 ermöglichen easy Identifizierung der Position der einfach ungesättigten Fettsäure-Doppelbindungen. 3-pyridylcarbinyl ester (Picolinyl ester) Derivate zur eindeutigen Identifizierung der Lage von Methyl ermöglichen verzweigten FAs 5. Kombination von chromatographischen Retention (ECL) und Massenspektren (DMOX und Picolinyl) Information erlaubt die Identifizierung der meisten FAs ohne die Notwendigkeit , komplexe Reinigungsverfahren zu verwenden, wie 1 für die Kernspinresonanz (NMR) -Spektrum, das unhinterfragbares Methode zur strukturellen Charakterisierung erforderlich . Bakterien der Gattung Bacillus, die einige menschliche und tierische Pathogene umfassen, können sehr unterschiedliche Nischen zu kolonisieren und werden daher in der Umwelt weit 6 verteilt. Unter der Gattung Bacillus, der Zusammensetzung FA durch die ökologische Nische der Spezies mit Modulationen in FA Muster beeinflusst zu einem breiten Spektrum von Umgebungsänderungen (zB Wachstumsmedium, Temperatur, anzupassenpH, etc.) 7-9. Aufgrund der relativen Homogenität des FA Muster über Spezies der Gattung Bacillus während des Wachstums in standardisierten Bedingungen, Bestimmung der FA Zusammensetzung ist eines der wesentlichen Kriterien verwendet , um die Bacillus - Arten definieren. Ein einzigartiges Merkmal der Gattung Bacillus ist die Fülle an verzweigtkettigen FAs enthält 12-17 Kohlenstoffe 10-12 mit dem Verhältnis zwischen iso und anteiso Isomere ein wichtiger Faktor für die Anpassung an Umweltbedingungen zu sein. Bacillus species anzupassen auch gegenüber Umweltschwankungen , indem der Anteil an ungesättigten Fettsäuren zu verändern. In einigen Arten, wie Bacillus cereus, zwei Fettsäure-Desaturasen schaffen Doppelbindungen in unterschiedlichen Positionen der Alkylkette 13 mit unterschiedlichen Rollen in Anpassung 9. Das Beispiel des Bacillus genus veranschaulicht die Bedeutung der eben die Doppelbindung Position zu identifizieren und FA Verzweigung. Sammelnhungsweise, hat Identifizierung von Bacillus FA Muster mehrere nützliche Anwendungen. Hier schlagen wir eine neue GC-MS - Ansatz für Bacillus FA Mustererkennung, die die inhärenten Grenzen eines klassischen GC-MS - Analyse überwindet. Dieser innovative Ansatz kann direkt auf rohen biologischen Material verwendet werden, und besteht aus einer Kombination von bestehenden Techniken: Informationen über die Retentionszeiten (ECL) und Massenspektren verschiedener FAs Derivate (FAME, DMOX und Picolinyl-Ester). Wir verwenden die folgende FA-Nomenklatur. i, a und n iso anzuzeigen, verzweigten anteiso Methyl und geradkettige Fettsäure, respectively. Ungesättigte FAs wurden durch C genannt: d, wobei C die Zahl der Kohlenstoffatome in der Fettsäure ist und d die Anzahl der Doppelbindungen ist. Δ x gibt die Position der Doppelbindung, wobei die Doppelbindung auf der x - ten Kohlenstoff-Kohlenstoff - Bindung befindet, von der Carbonsäure Ende zählen.

<table><tbody><tr><td>GC/MS</td><td>Shimadzu</td><td>QP2010</td><td></td></tr><tr><td>capillary column ZB WAX</td><td>Phenomenex</td><td>7HG-G007-11</td><td>30 m x 0.25 mm x 0.25&amp;nbsp;&amp;micro;m</td></tr><tr><td>Methanol Lichrosolv</td><td>VWR</td><td>1.06018.2500</td><td></td></tr><tr><td>potassium hydroxide</td><td>Aldrich</td><td>P1767</td><td></td></tr><tr><td>THF</td><td>Hipersolv Chromanorm</td><td>28559.320</td><td></td></tr><tr><td>Dichloromethane</td><td>Hipersolv Chromanorm</td><td>23373.320</td><td></td></tr><tr><td>Hexane</td><td>Hipersolv Chromanorm</td><td>24575.320</td><td></td></tr><tr><td>3-pyridinemethanol</td><td>Aldrich</td><td>P6-680-7</td><td></td></tr><tr><td>potassium tertiobutoxide</td><td>Aldrich</td><td>156671</td><td></td></tr><tr><td>2-amino-2-methyl-1-propanol</td><td></td><td>A-9879</td><td></td></tr><tr><td>MilliQ Academic</td><td>Millipore</td><td>ZMQS50001</td><td></td></tr><tr><td>Bacterial Acid Methyl Ester (BAME) Mix</td><td>Sigma-Aldrich</td><td>47080-U Supelco</td><td></td></tr></tbody></table>

identification of fatty acids in bacillus cereus

Die Bacillus - Arten enthalten Kette und ungesättigte Fettsäuren (FAs) mit unterschiedlichen Positionen der Methylverzweigung verzweigten (iso oder anteiso) und der Doppelbindung. Änderungen in FA Zusammensetzung, die eine entscheidende Rolle bei der Anpassung der Bakterien an ihre Umgebung zu spielen. Diese Modifikationen beinhalten eine Änderung in dem Verhältnis von iso gegen anteiso FAs verzweigt, und der Anteil an ungesättigten FAs relativ zu gesättigten FAs mit Doppelbindungen an spezifischen Positionen erstellt. Genaue Identifizierung des FA Profil ist notwendig , um die Anpassungsmechanismen von Bacillus - Spezies zu verstehen. Viele der FAs von Bacillus sind nicht im Handel erhältlich. Die vorgeschlagene Strategie identifiziert hierin FAs von Informationen über die Retentionszeit (Berechnung der äquivalenten Kettenlänge (ECL)) mit den Massenspektren von drei Arten von FA-Derivaten kombiniert: Fettsäuremethylester (FAME), 4,4-Dimethyl-oxazolin Derivate (DMOX) und3-pyridylcarbinyl Ester (Picolinyl). Diese Methode kann die FAs ohne die Notwendigkeit erkennen, das Unbekannte FAs zu reinigen. Vergleichen chromatographischen Profile von FAME hergestellt aus Bacillus cereus mit einem handelsüblichen Gemisch von Standards ermöglicht die Identifizierung von geradkettigen gesättigten FAs, die Berechnung der ECL, und Hypothesen über die Identität des anderen FAs. im Vergleich zu linearen gesättigten FAs mit der gleichen Anzahl von Kohlenstoffatomen FAMEs verzweigter gesättigter FAs, iso oder anteiso, in der ECL, eine konstante negative Verschiebung anzuzeigen. FAMEs ungesättigter FAs kann durch die Masse ihrer Molekülionen, und führen zu einer positiven Verschiebung der ECL im Vergleich zu den entsprechenden gesättigten FAs detektiert werden. Die Verzweigungsposition von Fas und die Doppelbindung Position von ungesättigten FAs kann durch die elektronen Ionisations-Massenspektren von Picolinyl und DMOX Derivate bzw. identifiziert werden. Dieser Ansatz identifiziert die unbekannten gesättigten Zweiged FAs, ungesättigte geradkettige FAs und ungesättigte verzweigte FAs aus dem cereus Extrakt B..

Die Strategie der FA Identifizierung von Bakterienzellen ist in 1 dargestellt. Jeder Schritt liefert ergänzende spektrale Information oder Informationen über chromatographische Retention. Schritt 1 besteht aus vorläufigen FA Identifizierung einer Standardlösung verwenden. Schritt 2 ermöglicht die Auslegung von FAME EI-Spektren und ihre ECL, um die Produkte zu vorläufig zu identifizieren. Schritt 3 identifiziert die genaue Abzweigstelle in verzweigtkettigen-FAs. Sch...

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Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

Identifizierung von Fettsäuren in<em&gt; Bacillus cereus</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

identification-of-fatty-acids-in-bacillus-cereus

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

9.5K Aufrufe.  INRA, Université d'Avignon. We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl). 

Serie: Identification of Fatty Acids in Bacillus cereus

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

Die Analyse der Fettsäuregehalt und Zusammensetzung in Mikroalgen

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Forschung

Umwelt

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Fettsäure- 13C Isotopologue Profilierung gibt Einblick in trophische Carbon Transfer und Fettstoffwechsel der Wirbellosen Verbraucher

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

Biochemie

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Isolierung von Lipoproteinpartikeln aus Hühnereigelb zur Untersuchung des bakteriellen Pathogens Fettsäure-Inkorporation in Membranphospholipide

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Optimierter Einbau von Alkynyl-Fettsäure-Analoga zum Nachweis von fettacylierten Proteinen mittels Klickchemie

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Analyse der neutralen Lipidsynthese in Saccharomyces cerevisiae durch metabolische Markierung und Dünnschichtchromatographie

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

Identifizierung antibakterieller Immunitätsproteine in Escherichia coli mittels MALDI-TOF-TOF-MS/MS und Top-Down-Proteomanalyse

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

Chemie

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Galle-Salz-induzierte Biofilmbildung im enterischen Krankheitserregern: Techniken für die Identifizierung und Quantifizierung

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Immunologie und Infektion

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Analyse der Lipidzusammensetzung von Mykobakterien mittels Dünnschichtchromatographie

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

Identifizierung von Fettsäuren in

Zusammenfassung

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Isolierung von Lipoproteinpartikeln aus Hühnereigelb zur Untersuchung des bakteriellen Pathogens Fettsäure-Inkorporation in Membranphospholipide

Quantitative Analyse des Zelllipidoms von Saccharomyces Cerevisiae mit Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie

Optimierter Einbau von Alkynyl-Fettsäure-Analoga zum Nachweis von fettacylierten Proteinen mittels Klickchemie

Analyse der neutralen Lipidsynthese in Saccharomyces cerevisiae durch metabolische Markierung und Dünnschichtchromatographie

Identifizierung antibakterieller Immunitätsproteine in Escherichia coli mittels MALDI-TOF-TOF-MS/MS und Top-Down-Proteomanalyse

Galle-Salz-induzierte Biofilmbildung im enterischen Krankheitserregern: Techniken für die Identifizierung und Quantifizierung

Analyse der Lipidzusammensetzung von Mykobakterien mittels Dünnschichtchromatographie

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs