The financial support of R. Menzel and L. Ruess by the Deutsche Forschungsgemeinschaft (RU RU780/11-1) is gratefully acknowledged. R. Nehring was funded by RU 780/10-1. Finally, we are extremely thankful to Dr. Hazel Ruvimbo Maboreke for proofreading our manuscript.

<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 profiling
A detailed analysis of the quantitative aspects in 13C distribution in FAs needs cutting-edge technology to assign carbon partitioning in food webs. The present work employed isotopologue profiling to assess the 13C/12C ratios in common FA biomarkers for tropic interactions. This method is well established for amino acid analysis by liquid chromatography (LC-MS) and was applied for investigations of carbon metabolism in pathogenic bacteria17,23. Only recently, was isotopologue profiling further developed as a tool to study dietary routing of lipids in small soil invertebrates by Menzel et al.24 Since this method is not hampered by the &#34;carry over&#34; of the 13C signal, it is therefore advantageous for the evaluation of organic tissue highly enriched with a specific isotope compared to GC-C-IRMS32.
With this approach, the mass-to-charge (m/z) values in the molecular ion and its isotopologues is determined by GC-MS using EI and SIM mode. In comparison to the full scan, the sensitivity in SIM can be increased by about factor 100, which even outstrips the GC-FID quantification range33. Further, by acquisition of a specified group of ions in a given time window, the analyte is measured reliably despite a background of co-eluting peaks30. Thurnhofer and Vetter34 proved that SIM is suitable for both FA identification and quantification in food samples even with low amounts of lipids available.
When using SIM for 13C isotopologue profiling at least two methodological constraints have to be considered. Firstly, molecular ions are of relatively low abundance and their isotopologues are even less. With a dirty ion source, these become less abundant and as a consequence the SIM method will only be semi-quantitative33. Secondly, the increased occurrence of 13C at one or more carbon positions of the FA decreases the 13C natural abundance of associated carbon atoms. These changes with labelling are non-linear, a phenomenon called &#34;skew&#34; in natural abundance, which can lead to underestimation of enrichment at these masses20. This skew effect must be accounted for either by using a correction matrix based on FAME standards (see Fernandez et al.) or by including natural controls, i.e., FAs from experimental organisms without label. The latter was performed in the present work comparing the same FA in labelled (day 1 and later) and non-labelled Collembola (day 0).
Dietary routing and 13C flux
While the methodological advantages of isotopologue profiling for 13C determination in FAs are obvious, in vivo metabolism of organisms can be hindered by biological means. Isotopic and metabolic steady state are difficult to achieve when, e.g., diet usage or the physiological status of consumers are not known, hampering obtaining absolute carbon fluxes. However, by comparing 13C isotopologues profiles of FAs in the diet and the consumer, the relative proportion of FA assimilation and metabolic pathways can be obtained. This approach allows following the specific fate of a marker FA based on their 13C label by tracing its dietary routing. MS-SIM demonstrated that the marker FA was mainly routed into the NFLA fractions of both Collembola species analyzed24. Moreover, the marker FA was stored in the neutral lipids with only minor or no modifications. Only elongation of the dietary 13C16:0 to 18:0 and desaturation to the monoenoic equivalents were detected (see Figure 4). The presence of the ion M+16 in the C16 and C18 FAs of day 1 samples confirms that these FAs are descendants of the supplemented entirely labeled palmitic acid. This finding underpins earlier studies that were based on the FA pattern of consumer and diet solely5,35 and suggests that marker FAs are incorporated as entire molecules into consumer tissue (for a review, see Ruess and Chamberlain8).
Meanwhile, the isotopologue 13C pattern of PLFAs revealed that the observed increase in 13C20:46 over time is due to both elongation/desaturation of entirely labeled precursor FAs and de novo synthesis with the latter also including 13C labeled acetyl-CoA as indicated by the presence of M+18 ion traces in C20 and also C18 FAs (see Figure 4). Figure 5 summarizes the dietary routing of supplemented 13C16:0 within PLFAs and NLFAs of the observed Collembola species. Interestingly, only 13C palmitic acid of PLFAs is subject to stronger changes, whereas the metabolic transformation in the NLFAs remained limited to only one elongation and two desaturation steps.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57110/57110fig5.jpg" /> 
Figure 5: Fate of dietary 13C palmitic acid in consumer lipids. The flow-charts show the proposed fate of incorporated 13C16:0 in the investigated Collembola species. Arrows symbolize the different levels of metabolic turnover with different thickness. <a href="/files/ftp_upload/57110/57110fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To summarize, isotopologue profiling by MS-SIM clearly shows that FA use as trophic marker is a robust and reliable method in the ecology of food webs. Although the experimental design did not allow the precise quantification of the 13C flux (e.g., as atom% 13C of the supplemented FA) for the different steps in the pathways of FA metabolism, isotopologue profiling is a useful tool for all ecological studies using fatty acids as trophic markers. A goal for the future is to obtain a quantitative consumer FA carbon budget by using closed microcosm systems and feeding consumers defined amounts of labeled precursors of fatty acids.

In a cryptic habitat such as soil, trophic relationships are difficult to address and are further restricted by the small size of the fauna. The last decade has seen advances in biochemical ecology, particularly in the use of fatty acids as biomarkers for defining feeding strategies of the soil fauna under field conditions1,2,3. This is based on the fact that fatty acids from resources can be incorporated in consumer tissue as entire molecules, a process termed dietary routing4. Transfer of fatty acids has been reported over three trophic levels, i.e., from fungi to nematodes to Collembola5. Recently, the predatory fauna was considered6,7 and the first reviews on fatty acids as trophic markers in soil food webs have been published8,9.
More detailed information on trophic interactions is attained by fatty acid stable isotope probing (FA-SIP). The determination of 13C/12C ratios in fatty acids in diets and consumers can ascribe binary links and estimate the associated carbon flow, and has been employed in terrestrial, fresh water, and marine food webs10,11,12,13. The basic assumption is that dietary routed fatty acids are not subject to enzymatic processes; therefore, their 13C signal, i.e., the 13C/12C ratio of the fatty acid, in the consumer is similar to that in the diet1. However, a gradual depletion of the 13C signature up the food chain has been reported in aquatic systems, thereby hindering broad application of FA-SIP in trophic studies14,15,16. Moreover, knowledge in the lipid metabolism in most invertebrates in terrestrial food webs is still limited.
An understanding of the lipid metabolism pathways in consumers is essential for the usage of trophic marker fatty acids as means for the determination of the quantitative carbon flow in food web ecology. With this in mind, 13C-isotopologue profiling, which in principle can be applied for investigation of the carbon metabolism of any biological system17, is a promising method. Following the introduction of a 13C-labelled carbon substrate, the distribution of the 13C in the metabolic network is traceable since the generated metabolic products in the consumer show a specific isotopologue distribution. This can be assessed by quantitative nuclear metabolic resonance spectroscopy18,19 or mass spectrometry20,21, with the latter favored in biological samples with low biomass due to its higher sensitivity.
Although isotopologue profiling has been successfully applied to amino acids and provided insight into the in vivo carbon metabolism of bacterial pathogens17,22,23, its implementation in fatty acids has lagged behind. The first detailed analysis on the fate of a stable isotope labelled precursor fatty acid, its dietary routing or degradation via &#946;-oxidation, in soil invertebrate consumers, was recently performed by Menzel et al.24. Here, the methodological basics for incorporation experiments with 13C labelled fatty acids followed by isotopologue analysis of key descendants in frequent soil invertebrates, the Collembola, are provided. These microarthropods are a good model group as they form important components of the soil food web and are well investigated for their trophic marker fatty acids8,25.
An understanding of the lipid metabolism pathways in consumers is essential for the usage of trophic marker fatty acids as means for the determination of the quantitative carbon flow in food web ecology. The present protocol gives the design and set up for a laboratory feeding experiment, and the biochemical procedures for extraction and methylation of dominant lipids fractions (neutral lipids, phospholipids) from Collembola. It demonstrates how the isotopologue composition of fatty acids is analyzed by mass spectrometry and describes the related formula and calculations. This procedure results in: (i) the ratios of isotopologues exceeding the mass of the parent ion (i.e., the fatty acid molecular ion M+) by one or more mass units (M+1, M+2, M+3, etc.) and (ii) the overall 13C enrichment in fatty acids derived from the 13C labelled precursor. Although used for Collembola, this approach can generally be applied to any other predator-prey interaction on the premise that these are culturable in sufficient quantity under controlled conditions to ensure a successful label uptake and subsequent verification.

<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

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.

Fresh weight and lipid content of Collembola 
In the course of the described experiment, the content in NLFAs and PLFAs did not change significantly over time, whereas the fresh weight of specimens increased slightly but not significantly24. Both parameters indicate a good level of physical fitness of the Collembola specimens. Be aware to investigate Collembola&#39;s fresh weight and lipid content throughout the experiment corresponding to the sampling days for fatty acid and isotope analysis. Note that a loss of weight and/or a decrease of lipid content during the experimental period indicate a reduced fitness of the test organism and the derived data drastically lose significance.
Isotopologue detection 
The special significance of isotopologue profiling projects lies in the individual detection and quantification of both the molecular ion and all isotopologues in a specific fatty acid. For example, in case of 16:0 the ions range from 270, i.e., molecular ion M+ (C skeleton mainly composed of 12C atoms) to 286 (isotopologue M+16 - carbon chain completely substituted with the heavy 13C). Figure 2A presents a SIM-MS spectrum of pure 16:0, palmitic acid. The natural abundance of 13C in this compound becomes detectable by the presence of M+1 and M+2 isotopologues in addition to the molecular ion (M+). By comparison, Figure 2B shows the spectrogram of the entirely labeled palmitic acid (99 atom% 13C) used in this study. Here, the sole occurrence of the ion M+16 reflects the high purity of this synthetically labeled compound.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57110/57110fig2.jpg" /> 
Figure 2: Representative SIM scans of pure (A) unlabeled and (B) entirely labeled palmitic acid. Note the natural abundance of M+1 and M+2 isotopologues in addition to the molecular ion (M+) in the unlabeled C 16:0 (A) but the exclusive presence of the M+16 isotopologue in the entirely labeled fatty acid (99 atom% 13C). <a href="/files/ftp_upload/57110/57110fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Elongation/desaturation versus de novo synthesis 
After harvesting the test organisms, lipid extraction and derivatization, the generated FAMEs are ready to be analyzed by GC and SIM-MS. By examination of isotopologue profiling data, one can now distinguish between de novo synthesis and desaturation/elongation events of the entire labeled marker fatty acid. The former can take place, at least partially, based on 13C Acetyl-CoA as product of the degradation of the labelled precursor via beta-oxidation. Figure 3 shows representative examples of SIM scans derived from four dominant fatty acids in the PLFA fraction of H. nitidus at the first sampling day after labelling. The assimilation of the labelled marker molecule into palmitic acid becomes visible by the abundance of the M+16 - isotopologue, as it represents the entirely labeled fatty acid (Figure 3A - 16:0). Further, desaturation of 16:0 (Figure 3B - 16:1&#969;7) or elongation plus desaturation (Figure 3C - 18:1&#969;9, Figure 3D - 20:4&#969;6) can be assigned. Thereby, the detection of isotopologues bigger than M+16, e.g. M+17 and M+18 demonstrate chain elongation of the labelled precursor 16:0 by the use of 13C2-fragments. De novo biosynthesis based on 13C Acetyl-CoA of fatty acids is indicated if isotopologues close to the molecular ion, i.e., M+1 to M+2 get significantly more frequent than in the control represented by day 0 sampling.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57110/57110fig3.jpg" /> 
Figure 3: Representative SIM (selected ion monitoring) scans of fragment ions in fatty acids of Heteromurus nitidus (PLFA fraction, 1 day after labelling). (A) Palmitic acid (16:0) (B) Palmitoleic acid (16:1&#969;7) (C) Oleic acid (18:1&#969;9), and (D) Arachidonic acid (20:4&#969;6). <a href="/files/ftp_upload/57110/57110fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Further illustrating this, Figure 4 compares the initial isotopologue pattern of the five most heavily labeled fatty acids (analyzed both in PLFAs and NLFAs); compared are the data from the Collembola species P. fimata on day 0 and day 1. On day 1, the 13C signal in C16 and C18 FAs was almost entirely based on the occurrence of the M+16molecule ion, resulting from entirely labeled 13C 16:0, supplemented to the food. As mentioned above, the observed traces of M+18 of 18:0 and 18:1&#969;9 suggest the introduction of a minor portion of 13C Acetyl-CoA via chain elongation from 16:0 to 18:0. For 18:1&#969;9 this was more pronounced in the PLFA faction. Such 13C acetyl-CoA derives from &#946;-oxidation of labeled 13C 16:0 molecules. Incorporation of 13C acetyl-CoA also occurred in the elongation step from chain length C18 to C20, as assigned by the 20:4&#969;6 of the PLFA samples. Thereby, the 13C enrichment in M+16 and M+18 significantly increased with time until day 14 (Figure 4). Moreover, M+2 of this PLFA 20:4&#969;6 at day 14 increased when compared to day 0 or 1. This 13C allocation in the different isotopologues indicates that the formation of 20:4&#969;6 is based both on de novo synthesis with inclusion of acetyl-CoA labeled with 13C or at natural 13C abundance and the elongation/desaturation of the entire 13C16:0 precursor molecule. In contrast, distinctly labeled 20:4&#969;6 did not appear in the NLFA fraction (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57110/57110fig4.jpg" /> 
Figure 4: Isotopologue pattern of the 13C incorporation by SIM (selected ion monitoring). Presented are the relative portions of the ions M+1 to M+3 and M+16 to M+20 of the five most heavily labeled NLFAs and PLFAs from Protaphorura fimata, estimated on day 0 and day 1. Only for 20:4&#969;6 the day 14 data are presented in addition. P&#60;0.05 (Dunnett&#39;s test with day 0 data used as reference). This figure is a reprint of Figure 5 published in Menzel et al.24 <a href="/files/ftp_upload/57110/57110fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.9K Views.  Humboldt-Universität zu Berlin. 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.

Video: 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).

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ...