Die finanzielle Unterstützung von R. Menzel und L. Ruess von der Deutschen Forschungsgemeinschaft (RU RU780/11-1) ist dankbar anerkannt. R. Nehring wurde von RU 780/10-1 finanziert. Schließlich sind wir sehr dankbar, dass Dr. Hazel Ruvimbo Maboreke für das Korrekturlesen unserer Handschrift.

<ol>
	<li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+lipid+analysis+to+understand+trophic+interactions+in+soil.">Application of lipid analysis to understand trophic interactions in soil.</a> Ecology. 86 (8), 2075-2082 (2005).</li><li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+composition+of+Collembola+and+their+food+resources+in+deciduous+forest+stands+-+Implications+for+feeding+strategies.">Lipid composition of Collembola and their food resources in deciduous forest stands - Implications for feeding strategies.</a> Soil Biology and Biochemistry. 39 (8), 1990-2000 (1990).</li><li>Chamberlain, P. M., Bull, I. D., Black, H. I. J., Ineson, P., Evershed, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+and+change+in+Collembola+fed+differing+diets:+identification+of+trophic+biomarkers.">Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers.</a> Soil Biology and Biochemistry. 37 (9), 1608-1624 (2005).</li><li>Stott, A. W., Davies, E., Evershed, R. P., Tuross, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+routing+of+dietary+and+biosynthesised+lipids+through+compound-specific+stable+isotope+(delta+C-13)+measurements+at+natural+abundance.">Monitoring the routing of dietary and biosynthesised lipids through compound-specific stable isotope (delta C-13) measurements at natural abundance.</a> Naturwissenschaften. 84 (2), 82-86 (1997).</li><li>Ruess, L., Haggblom, M. M., Langel, R., Scheu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+isotope+ratios+and+fatty+acid+composition+as+indicators+of+animal+diets+in+belowground+systems.">Nitrogen isotope ratios and fatty acid composition as indicators of animal diets in belowground systems.</a> Oecologia. 139 (3), 336-346 (2004).</li><li>Pollierer, M. M., Scheu, S., Haubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+it+to+the+next+level:+Trophic+transfer+of+marker+fatty+acids+from+basal+resource+to+predators.">Taking it to the next level: Trophic transfer of marker fatty acids from basal resource to predators.</a> Soil Biology and Biochemistry. 42 (6), 919-925 (2010).</li><li>Ferlian, O., Scheu, S., Pollierer, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+interactions+in+centipedes+(Chilopoda,+Myriapoda)+as+indicated+by+fatty+acid+patterns:+Variations+with+life+stage,+forest+age+and+season.">Trophic interactions in centipedes (Chilopoda, Myriapoda) as indicated by fatty acid patterns: Variations with life stage, forest age and season.</a> Soil Biology and Biochemistry. 52, 33-42 (2012).</li><li>Ruess, L., Chamberlain, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fat+that+matters:+Soil+food+web+analysis+using+fatty+acids+and+their+carbon+stable+isotope+signature.">The fat that matters: Soil food web analysis using fatty acids and their carbon stable isotope signature.</a> Soil Biology and Biochemistry. 42 (11), 1898-1910 (2010).</li><li>Traugott, M., Kamenova, S., Ruess, L., Seeber, J., Plantegenest, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirically+characterising+trophic+networks:+What+emerging+DNA-based+methods,+stable+isotope+and+fatty+acid+analyses+can+offer.">Empirically characterising trophic networks: What emerging DNA-based methods, stable isotope and fatty acid analyses can offer.</a> Adv Ecol Res. 49, 177-224 (2013).</li><li>Hammer, B. T., Fogel, M. L., Hoering, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+carbon+isotope+ratios+of+fatty+acids+in+seagrass+and+redhead+ducks.">Stable carbon isotope ratios of fatty acids in seagrass and redhead ducks.</a> Chemical Geology. 152 (1-2), 29-41 (1998).</li><li>Budge, S. M., Iverson, S. J., Koopman, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+trophic+ecology+in+marine+ecosystems+using+fatty+acids:+A+primer+on+analysis+and+interpretation.">Studying trophic ecology in marine ecosystems using fatty acids: A primer on analysis and interpretation.</a> Marine Mammal Science. 22 (4), 759-801 (2006).</li><li>Haubert, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+structure+and+major+trophic+links+in+conventional+versus+organic+farming+systems+as+indicated+by+carbon+stable+isotope+ratios+of+fatty+acids.">Trophic structure and major trophic links in conventional versus organic farming systems as indicated by carbon stable isotope ratios of fatty acids.</a> Oikos. 118 (10), 1579-1589 (2009).</li><li>Ngosong, C., Raupp, J., Richnow, H. H., Ruess, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Collembola+feeding+strategies+by+the+natural+13C+signal+of+fatty+acids+in+an+arable+soil+with+different+fertilizer+regimes.">Tracking Collembola feeding strategies by the natural 13C signal of fatty acids in an arable soil with different fertilizer regimes.</a> Pedobiologia. 54 (4), 225-233 (2011).</li><li>Bec, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+reliability+of+fatty+acid-specific+stable+isotope+analysis+for+trophic+studies.">Assessing the reliability of fatty acid-specific stable isotope analysis for trophic studies.</a> Methods in Ecology and Evolution. 2 (6), 651-659 (2011).</li><li>Gladyshev, M. I., Makhutova, O. N., Kravchuk, E. S., Anishchenko, O. V., Sushchik, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+fractionation+of+fatty+acids+of+Daphnia+fed+laboratory+cultures+of+microalgae.">Stable isotope fractionation of fatty acids of Daphnia fed laboratory cultures of microalgae.</a> Limnologica. 56 (Supplement C. 56 (Supplement C), 23-29 (2016).</li><li>Gladyshev, M. I., Sushchik, N. N., Kalachova, G. S., Makhutova, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+composition+of+fatty+acids+in+organisms+of+different+trophic+levels+in+the+Yenisei+river.">Stable isotope composition of fatty acids in organisms of different trophic levels in the Yenisei river.</a> PLoS One. 7 (3), e34059 (2012).</li><li>Eisenreich, W., Dandekar, T., Heesemann, J., Goebel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+metabolism+of+intracellular+bacterial+pathogens+and+possible+links+to+virulence.">Carbon metabolism of intracellular bacterial pathogens and possible links to virulence.</a> Nature Reviews Microbiology. 8 (6), 401-412 (2010).</li><li>Eylert, E., Bacher, A., Eisenreich, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR-based+isotopologue+profiling+of+microbial+carotenoids.">NMR-based isotopologue profiling of microbial carotenoids.</a> Methods Mol Biol. 892, 315-333 (2012).</li><li>Garton, N. J., O'Hare, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis:+feeding+the+enemy.">Tuberculosis: feeding the enemy.</a> Chemical Biology. 20 (8), 971-972 (2013).</li><li>Rosenblatt, J., Chinkes, D., Wolfe, M., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+tracer+analysis+by+GC-MS,+including+quantification+of+isotopomer+effects.">Stable isotope tracer analysis by GC-MS, including quantification of isotopomer effects.</a> Am J Physiol. 263 (3), E584-E596 (1992).</li><li>Fernandez, C. A., Des Rosiers, C., Previs, S. F., David, F., Brunengraber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+13C+mass+isotopomer+distributions+for+natural+stable+isotope+abundance.">Correction of 13C mass isotopomer distributions for natural stable isotope abundance.</a> J Mass Spectrom. 31 (3), 255-262 (1996).</li><li>Heuner, K., Eisenreich, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+metabolism+of+legionella+by+isotopologue+profiling.">The intracellular metabolism of legionella by isotopologue profiling.</a> Methods Mol Biol. 954, 163-181 (2013).</li><li>Willenborg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+pivotal+carbon+metabolism+of+Streptococcus+suis+serotype+2+under+ex+vivo+and+chemically+defined+in+vitro+conditions+by+isotopologue+profiling.">Characterization of the pivotal carbon metabolism of Streptococcus suis serotype 2 under ex vivo and chemically defined in vitro conditions by isotopologue profiling.</a> J Biol Chem. 290 (9), 5840-5854 (2015).</li><li>Menzel, R., Ngosong, C., Ruess, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopologue+profiling+enables+insights+into+dietary+routing+and+metabolism+of+trophic+biomarker+fatty+acids.">Isotopologue profiling enables insights into dietary routing and metabolism of trophic biomarker fatty acids.</a> Chemoecology. 27 (3), 101-114 (2017).</li><li>Buse, T., Ruess, L., Filser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+trophic+biomarkers+for+Collembola+reared+on+algal+diets.">New trophic biomarkers for Collembola reared on algal diets.</a> Pedobiologia. 56 (3), 153-159 (2013).</li><li>Hutson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+variations+of+the+plaster-charcoal+culture+method+on+a+Collembolan,+Folsomia+candida.">Effects of variations of the plaster-charcoal culture method on a Collembolan, Folsomia candida.</a> Pedobiologia. 18, 138-144 (1978).</li><li>Fountain, M. T., Hopkin, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folsomia+candida+(Collembola):+a+&amp;#34;standard&amp;#34;+soil+arthropod.">Folsomia candida (Collembola): a &amp;#34;standard&amp;#34; soil arthropod.</a> Annu Rev Entomol. 50, 201-222 (2005).</li><li>ISO, I. O. f. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soil Quality-Inhibition of reproduction of Collembola (Folsomia candida) by soil pollutants. , (1999).</li><li>Welch, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+cellular+fatty+acid+analysis.">Applications of cellular fatty acid analysis.</a> Clin Microbiol Rev. 4 (4), 422-438 (1991).</li><li>Dodds, E. D., McCoy, M. R., Rea, L. D., Kennish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+quantification+of+fatty+acid+methyl+esters:+flame+ionization+detection+vs.+electron+impact+mass+spectrometry.">Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry.</a> Lipids. 40 (4), 419-428 (2005).</li><li>Kuppardt, S., Chatzinotas, A., Kastner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+fatty+acid+and+RNA+stable+isotope+probing-based+method+for+tracking+protist+grazing+on+bacteria+in+wastewater.">Development of a fatty acid and RNA stable isotope probing-based method for tracking protist grazing on bacteria in wastewater.</a> Appl Environ Microbiol. 76 (24), 8222-8230 (2010).</li><li>Zhang, X., He, H., Amelung, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GC/MS+method+for+the+assessment+of+15N+and+13C+incorporation+into+soil+amino+acid+enantiomers.">A GC/MS method for the assessment of 15N and 13C incorporation into soil amino acid enantiomers.</a> Soil Biology and Biochemistry. 39 (11), 2785-2796 (2007).</li><li>Vetter, W., Thurnhofer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fatty+acids+by+mass+spectrometry+in+the+selected+ion+monitoring+mode.">Analysis of fatty acids by mass spectrometry in the selected ion monitoring mode.</a> Lipid Technol. 19 (8), 184-186 (2007).</li><li>Thurnhofer, S., Vetter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gas+chromatography/electron+ionization-mass+spectrometry-selected+ion+monitoring+method+for+determining+the+fatty+acid+pattern+in+food+after+formation+of+fatty+acid+methyl+esters.">A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters.</a> J Agric Food Chem. 53 (23), 8896-8903 (2005).</li><li>Haubert, D., Haggblom, M. M., Scheu, S., Ruess, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fungal+food+quality+and+starvation+on+the+fatty+acid+composition+of+Protaphorura+fimata+(Collembola).">Effects of fungal food quality and starvation on the fatty acid composition of Protaphorura fimata (Collembola).</a> Comparative Biochemistry and Physiology B-Biochemistry &amp; Molecular Biology. 138 (1), 41-52 (2004).</li></ol>

Isotopologue Profilierung
Eine detaillierte Analyse der quantitativen Aspekte bei 13C Verteilung in FAs braucht Spitzentechnologie, Kohlenstoff in Nahrungsketten Partitionierung zuzuweisen. Die vorliegende Arbeit beschäftigt Isotopologue Profilierung zur Beurteilung der 13C /12C-Verhältnisse in gemeinsamen FA Biomarker für tropische Interaktionen. Diese Methode ist gut etabliert für Aminosäureanalyse von Flüssigchromatographie (LC-MS) und...

In einem kryptischen Lebensraum wie Boden trophische Beziehungen sind schwer zu Adresse und sind durch die geringe Größe der Fauna weiter beschränkt. Im letzte Jahrzehnt hat Fortschritte in biochemische Ökologie, insbesondere bei der Verwendung von Fettsäuren als Biomarker für die Definition von Fütter Strategien der Bodenfauna unter Feld Bedingungen1,2,3gesehen. Dies ist, dass Fettsäuren aus Ressourcen in Verbraucher-Gewebe als gesamte Moleküle eingebaut werden können, bezeichnet ein Prozess diätetische routing4. Übertragung von Fettsäuren ist über drei trophischen Ebenen, d. h. aus Pilzen, Nematoden, Collembolen5berichtet worden. Vor kurzem, die räuberischen Fauna galt6,7 und die ersten Reviews auf Fettsäuren als trophische Markierungen im Boden Nahrungsnetze wurden veröffentlicht8,9.
Nähere Informationen zu trophischen Interaktionen wird durch Fettsäure-stabiler Isotope probing (FA-SIP) erreicht. Die Bestimmung von 13C /12C-Verhältnisse in Fettsäuren in Diäten und Verbraucher können zuschreiben binäre Verknüpfungen und Abschätzung den damit verbundenen CO2-Fluss und beschäftigt in terrestrischen, Süßwasser und marinen Nahrungsnetze10,11 ,12,13. Die Grundannahme ist, dass weitergeleitete diätetischen Fettsäuren nicht enzymatischen Prozessen unterworfen sind; daher ihre 13C signal, d. h. 13C /12C Verhältnis der Fettsäure in der Konsumgüterindustrie ist ähnlich wie in der Diät-1. Jedoch wurde eine allmähliche Entleerung der 13C Signatur oben in der Nahrungskette in aquatischen Systemen, dadurch behindern breite Anwendung des FA-SIP trophische Studien14,15,16berichtet. Kenntnisse in den Lipidstoffwechsel in den meisten Wirbellosen in terrestrischen Nahrungsketten ist übrigens nach wie vor begrenzt.
Das Verständnis der Lipid Metabolismus Bahnen bei den Verbrauchern ist Voraussetzung für die Nutzung der trophischen Marker Fettsäuren als Mittel zur Bestimmung der quantitativen Kohlenstoff Strömung im Nahrungsnetz Ökologie. Mit diesem im Verstand, 13C-Isotopologue Profilierung, welche im Prinzip für die Untersuchung von Kohlenstoff-Stoffwechsel jedes biologische System17, angewendet werden kann ist eine vielversprechende Methode. Nach der Einführung des 13C gekennzeichnet Carbon Substrat, die Verteilung der 13C in der metabolischen Netzwerkes ist seit der erzeugten Stoffwechselprodukte in der Consumer-Show eine spezifische Isotopologue Verteilung nachvollziehbar. Dies kann durch quantitative nukleare metabolische Resonanz Spektroskopie18,19 oder Massenspektrometrie20,21, mit der letzteren bevorzugten in biologischen Proben mit niedrigen Biomasse aufgrund seiner höheren beurteilt werden Empfindlichkeit.
Obwohl Isotopologue Profilierung wurde erfolgreich angewendet, Aminosäuren und bot Einblick in den in-Vivo Kohlenstoff-Stoffwechsel der bakteriellen Krankheitserregern17,22,23, deren Umsetzung in Fettsäuren Säuren ist zurückgeblieben. Die erste detaillierte Analyse über das Schicksal von einem stabilen isotopenmarkierter Vorläufer Fettsäure, diätetische routing oder Abbau durch β-Oxidation, Boden Wirbellosen Verbraucher, wurde vor kurzem von Menzel Et Al. durchgeführt. 24. hier werden die methodischen Grundlagen für Einbau Experimente mit 13C gekennzeichnet, gefolgt von Isotopologue Analyse der wichtigsten Nachkommen in häufigen Boden Wirbellosen, die Collembolen Fettsäuren zur Verfügung gestellt. Diese Microarthropods sind ein gutes Modell-Gruppe, da sie wichtige Bestandteile des Bodens Nahrungsnetz und sind gut für ihre trophische Marker Fettsäuren8,25untersucht bilden.
Das Verständnis der Lipid Metabolismus Bahnen bei den Verbrauchern ist Voraussetzung für die Nutzung der trophischen Marker Fettsäuren als Mittel zur Bestimmung der quantitativen Kohlenstoff Strömung im Nahrungsnetz Ökologie. Dieses Protokoll bietet das Design und die Einrichtung für ein Labor Ernährung Experiment und die biochemische Verfahren zur Extraktion und Methylierung von dominanten Lipide Brüche (neutralen Lipiden, Phospholipiden) von Collembolen. Es zeigt, wie die Isotopologue Zusammensetzung der Fettsäuren durch Massenspektrometrie analysiert wird und beschreibt die zugehörige Formel und Berechnungen. Dieses Verfahren führt zu: (i) die Verhältnisse der Isotopologues überschreiten die Masse des Ions (d. h. die Fettsäure Molekulare Ionen M+) Eltern von einem oder mehreren mass-Einheiten (M+ 1M+ 2M+ 3, etc.) und (Ii) die gesamte 13 C-Anreicherung in Fettsäuren abgeleitet aus den 13C gekennzeichneten Vorläufer. Obwohl für Collembolen verwendet, kann dieser Ansatz in der Regel auf andere Räuber-Beute-Interaktion auf der Prämisse angewendet werden, dass diese sind in ausreichender Menge unter kontrollierten Bedingungen zu eine erfolgreichen Label Akzeptanz zu gewährleisten und anschließende Überprüfung.

<table>
	<tbody>
		<tr>
			<td>neoLab-Round jars</td>
			<td>neoLab</td>
			<td>2-1506</td>
			<td>69 x 40 mm, 10 pacs/pack</td>
		</tr>
		<tr>
			<td>Charcoal activated</td>
			<td>Carl Roth</td>
			<td>X865.1</td>
			<td>p.a., powder, CAS No. 7440-44-0</td>
		</tr>
		<tr>
			<td>Alabaster Dental</td>
			<td>R&#214;HRICH-GIPSE</td>
			<td>---</td>
			<td>http://www.roehrich-gipse.de/dentalgipse.php</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Carl Roth</td>
			<td>7331.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>P717.1</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>Hexan</td>
			<td>Carl Roth</td>
			<td>7339.1</td>
			<td>HPLC &#8805; 98 %</td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether (MTBE)</td>
			<td>Carl Roth</td>
			<td>T175.1</td>
			<td>HPLC &#8805; 99,5 %</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Carl Roth</td>
			<td>7328.2</td>
			<td>HPLC &#8805; 99,9 %</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth</td>
			<td>6771.1</td>
			<td>p.a. &#8805;99 %, in pellets</td>
		</tr>
		<tr>
			<td>di-Natriumhydrogenphosphat</td>
			<td>Carl Roth</td>
			<td>P030.1</td>
			<td>p.a. &#8805;99 % , water free</td>
		</tr>
		<tr>
			<td>Na-dihydrogenphosphat Dihydrat</td>
			<td>Carl Roth</td>
			<td>T879.1</td>
			<td>p.a. &#8805;99 %</td>
		</tr>
		<tr>
			<td>Hypochloric acid (6 N)</td>
			<td>VWR International</td>
			<td>26,115,000</td>
			<td>AVS TITRINORM vol. solution</td>
		</tr>
		<tr>
			<td>Bond Elut (Columns)</td>
			<td>Agilent Tech.</td>
			<td>14102037</td>
			<td>HF Bond Elut-SI, 500 mg, 3 mL, 50/PK</td>
		</tr>
		<tr>
			<td>Pr&#228;paratengl&#228;ser Duran</td>
			<td>Glasger&#228;tebau Ochs</td>
			<td>135215</td>
			<td>&#216; 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket</td>
		</tr>
		<tr>
			<td>Supelco 37 Component FAME Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47885-U Supelco</td>
			<td>10&#160;mg/mL in methylene chloride, analytical standard</td>
		</tr>
		<tr>
			<td>FlowMesh</td>
			<td>Carl Roth</td>
			<td>2796.1</td>
			<td>Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing</td>
		</tr>
		<tr>
			<td>Bacterial Acid Methyl Ester (BAME) Mix</td>
			<td>Sigma-Aldrich</td>
			<td>47080-U Supelco</td>
			<td>10&#160;mg/mL in methyl caproate, analytical standard</td>
		</tr>
		<tr>
			<td>Methyl nonadecanoate</td>
			<td>Sigma-Aldrich</td>
			<td>74208</td>
			<td>analytical standard &#8805; 98.0 %</td>
		</tr>
		<tr>
			<td>Hexadecanoic acid-1-13C (Palmitic)</td>
			<td>Larodan Fine Chemicals</td>
			<td>78-1600</td>
			<td>GC &#8805; 98.0 % (13C: 99.0 %)</td>
		</tr>
		<tr>
			<td>RVC 2-25 CDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Compact benchtop midi concentrator</td>
		</tr>
		<tr>
			<td>Alpha 2-4 LDplus</td>
			<td>Martin Christ Gefrier-trocknungsanlagen</td>
			<td></td>
			<td>Drying manifold</td>
		</tr>
		<tr>
			<td>MZ 2C NT</td>
			<td>Vacuubrand GMBH</td>
			<td></td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Roto-Shake Genie</td>
			<td>Scientific Industries</td>
			<td></td>
			<td>Combined rocking and rotating device</td>
		</tr>
		<tr>
			<td>XP64 Micro Comparator</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>Super high precision balance</td>
		</tr>
		<tr>
			<td>GC-System 7890A</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Gas chromatograph</td>
		</tr>
		<tr>
			<td>7000 GC/MS Triple Quad</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Triple Quad mass spectrometer</td>
		</tr>
		<tr>
			<td>7683B Series Injector</td>
			<td>Agilent Tech.</td>
			<td></td>
			<td>Sample injector</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Centrifuge with 10 ml tube rotor</td>
		</tr>
	</tbody>
</table>

fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

Fettsäuren (FAs) sind nützliche Biomarker im Nahrungsnetz Ökologie, denn sie sind in der Regel als vollständige Molekül assimiliert und in Verbraucher-Gewebe mit Minderjährigen oder keine Änderung, so dass die diätetische routing zwischen verschiedenen trophischen Ebenen übertragen. Der FA-trophische Marker-Ansatz ist allerdings immer noch durch die begrenzte Kenntnisse in Lipidstoffwechsel von der Bodenfauna behindert. Diese Studie verwendete völlig gekennzeichneten Palmitinsäure (13C16:0, 99 % der Atom) als Tracer in Fettsäure-Stoffwechsel Bahnen von zwei weit verbreitete Boden Collembolen, Protaphorura Fimata und Heteromurus Redwood. Um das Schicksal und die metabolischen Veränderungen dieser Vorstufe zu untersuchen, ist eine Methode des Isotopologue profiling vorgestellt, durch Massenspektrometrie mit dem einzelnen Ion monitoring durchgeführt. Darüber hinaus wird die vorgelagerten Labor Fütterungsversuch sowie die Extraktion und die Methylierung von dominanten Lipid Brüche (neutralen Lipiden, Phospholipiden) und die zugehörige Formel und Berechnungen beschrieben. Nicht nur Ausbeute 13C beschriftet Vorläufer die insgesamt 13C-Anreicherung in Fettsäuren abgeleitet sondern produziert auch das Muster der Isotopologues überschreiten die Masse des Ions (d. h. das FA molekularen Ion Elternteil Isotopologue Profilierung M+) jeweils mit der Bezeichnung FA durch eine oder mehrere Masseneinheiten (M+ 1M+ 2M+ 3, etc.). Dieses Wissen erlaubt Rückschlüsse auf das Verhältnis der diätetischen routing von einem völlig verbrauchte FA im Vergleich zu de-Novo -Biosynthese. Die Isotopologue Profilierung empfiehlt sich als ein nützliches Instrument zur Bewertung der Fettsäure-Stoffwechsel in Bodentiere trophische Interaktionen zu entwirren.

Frische Gewicht und Lipid Inhalt der Collembolen Im Laufe des beschriebenen Versuchs änderte der Inhalt in NLFAs und PLFAs deutlich im Laufe der Zeit sich nicht während das Frischgewicht der Proben leicht aber nicht signifikant24erhöht. Beide Parameter zeigen ein hohes Maß an körperlicher Fitness der Collembolen Exemplare. Achten Sie darauf, zu untersuchen, Collembolens frische Gewicht und Lipid Inhalt während des Experiments, die Probenah...

Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

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

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

Die Fettsäure trophische Marker Ansatz, d. h. die Aufnahme von Fettsäuren als gesamte Molekül und Transfer in das Gewebe der Verbraucher ohne oder geringfügige Änderung, durch Wissenslücken im Fettsäure-Stoffwechsel der kleinen Boden Wirbellosen behindert wird. Isotopologue Profilierung dient als ein wertvolles Instrument zur trophische Interaktionen zu entwirren.

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

fatty-acid-13c-isotopologue-profiling-provides-insight-into-trophic

Research

JoVE Journal

Biochemistry

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

7.9K Aufrufe.  Humboldt-Universität zu Berlin. Die Fettsäure trophische Marker Ansatz, d. h. die Aufnahme von Fettsäuren als gesamte Molekül und Transfer in das Gewebe der Verbraucher ohne oder geringfügige Änderung, durch Wissenslücken im Fettsäure-Stoffwechsel der kleinen Boden Wirbellosen behindert wird. Isotopologue Profilierung dient als ein wertvolles Instrument zur trophische Interaktionen zu entwirren.

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

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

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 ...

Forschung

Biochemie

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Waschmittel-freie ultraschnelle Rekonstitution von Membranproteinen in Lipid Bilayer mit Fusogenic ergänzende Rechnung Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Polysome Profilerstellung in Leishmaniose, menschliche Zellen und Maus Hoden

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Probenahme und Vorbehandlung des Zahnschmelzes Carbonat für stabile Kohlenstoff und Sauerstoff-Isotopen-Analyse

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Nitropeptid Profilierung und Identifikation illustriert von Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

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 ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Waschmittelunterstützte Rekonstitution von rekombinantem Drosophila Atlastin in Liposomen für Lipid-Mischtests

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Profilierung von Ubiquitin und Ubiquitin-ähnlichen abhängigen posttranslationalen Änderungen und Identifizierung signifikanter Änderungen

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Einsatz der mikroskaligen Thermophorese zur Messung von Protein-Lipid-Wechselwirkungen

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Entfernung und Ersatz endogener Liganden aus lipidgebundenen Proteinen und Allergenen

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

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

Zusammenfassung

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