The overall goal of this isotopologue profiling is to follow the trophic transfer of dietary fatty acids in invertebrate consumers, including their processing and lipid metabolism, such as chain elongation or beta oxidation. This method can help answer key question in food web ecology, such as who's eating whom, a question not easy to answer in a cryptic ecosystem, such as soil. Demonstrating the technique will be Dilara Simsek, a master student from the lab of Professor Liliane Ruess.
To prepare plastic microcosms with tight-fitting lids, in a plaster pot, mix nine parts of plaster of Paris and one part of dry activated charcoal. Add 10 parts of distilled water, and allow the mixture to sit at room temperature without stirring for five minutes. Use a lab spoon to stir the mixture in a clockwise direction at moderate speed to avoid air bubbles until a thick, soupy consistency is achieved.
Then, pour the mixture into the microcosms to a height of about one centimeter. Smooth the plaster by gentle tapping on the bench and swirling. Note that holes from air bubbles and furrows, added with a sterile spatula, may encourage fertile Collembola to lay eggs there.
However, this study avoided holes and furrows in favor of having the same reproducible conditions. After allowing the microcosms to dry and then moistening them, use a suction tube fitted with a small piece of mesh to transfer 30 freshly hatched Collembola to the plaster base of the microcosms. Provide about a knife tip's worth of granulated dry Baker's yeast as food, and renew it at least twice per week.
Plan three independent replicates per sampling day. In this study, days zero to seven and 14 were sampled. Incubate the microcosms in the dark at 15 degrees Celsius, and maintain a constant temperature, as fatty acids are altered by animal metabolism to meet the requirements for membrane fluidity.
To harvest animals, directly after weighing according to the text protocol, carefully transfer the animals from the scale pans into 10-milliliter glass test tubes. Use one milliliter of HPLC-grade methanol to fill the tubes, and store them at negative 20 degrees Celsius until analysis. After preparing three glass test tubes for each batch of samples and reducing the methanol applied to the samples for storage, add five milliliters of single-phase extraction solvent to each sample, including blanks, and extract the Collembolan lipids at room temperature by shaking at 200 rpm overnight.
After extracting the samples according to the text protocol, to carry out fatty acid pattern analysis, precondition a silica acid column for each sample by adding two milliliters of chloroform to each column. After the chloroform has passed through the columns, use a glass Pasteur pipette to remove the lower chloroform phase from the samples. Then, transfer it to an individual column.
Use five milliliters of chloroform to elute the neutral lipids, or NLFAs, in fractions into individual glass vessels. Then, use 10 milliliters of acetone to elute the glycolipids and five milliliters of methanol to elute the phospholipid fatty acids, or PLFAs. Following extraction, transfer the NLFA and PLFA open tubes to a rotational vacuum concentrator, or RVC, and evaporate the samples at 60 degrees Celsius in a vacuum of 24 hPa for approximately 90 minutes or until dry.
After isolating and washing the fatty acid methyl esters, or FAMEs, use a glass Pasteur pipette to transfer the upper lipid-containing phase to a glass chromatography sample vial equipped with a Teflon septum. Encapsulate the vial, and store it at negative 20 degrees Celsius until analysis. To identify FAMEs in NFLA and PLFA samples, on a GC-FID, set up a sequence with the respective software of the instrument according to the manufacturer's instructions.
Start the sequence with the external standard mixtures, followed by the samples. There will be slight delay in elution time of the fatty acids from the GC column with each run. Either include a standard every 10th run in the sample sequence, or use retention time locking for palmitic acid.
Set the program, and calculate the nmol of fatty acid according to the text protocol. To carry out carbon-13 analysis by isotopologue profiling, use a polar capillary column, as it further allows the separation of unsaturated fatty acids, even with the same number of double bonds. The choice of the GC column is critical for the results, as it determines a good representation of the molecule ion in the fatty acids.
For a DB-23 column, start oven temperature at 130 degrees Celsius, and increase it by 6.5 degrees Celsius per minute to 170 degrees Celsius. Follow with an increase of three degrees Celsius per minute to 203 degrees Celsius, and hold for 1.9 minutes. Then, proceed with an increase of 40 degrees Celsius per minute to 230 degrees Celsius, and hold for 8.3 minutes.
Set the transfer line temperature to 280 degrees Celsius. After completing the program setup according to the text protocol, determine carbon-13 incorporation in the molecular ion of fatty acids by first running an initial scan to see what is present. Then, run selected ion monitoring, or SIM, on the appropriate ions, and acquire data at molecular masses of interest by selecting m/z scan windows, or SIM groups, encompassing the chromatographic peak time of the respective fatty acid.
Typically, monitor two to four ions per analyte and time window. For mass development, we use total ion counts with external fatty acid standards. Based on their retention time and fragment ions, time segments are chosen.
Detect the molecular ion, M plus, of the respective fatty acid and all its isotopologues, M plus one, M plus two, et cetera. Assign trophic carbon flux, the position of carbon-13 incorporation, and calculate carbon-13 enrichment according to the text protocol. This figure presents a SIM-MS spectrum of pure 16-to-zero palmitic acid.
The natural abundance of carbon-13 in this compound becomes detectable by the presence of M plus one and M plus two isotopologues in addition to the molecular ion M plus. By comparison, this spectrogram represents the entire labeled palmitic acid. Here, the sole occurrence of the ion M plus 16 reflects the high purity of the synthetically labeled compound.
The following SIM scans are derived from four dominant fatty acids in the PLFA fraction of Heteromurus nitidus at the first sampling day after labeling. The assimilation of the labeled marker molecule into palmitic acid becomes visible by the abundance of the M plus 16 isotopologue, as it represents the entire labeled fatty acid. Furthermore, desaturation of 16 to zero or elongation plus desaturation can be assigned.
Thereby, the detection of isotopologues bigger than M plus 16, for example, M plus 17 and M plus 18, demonstrate chain elongation of the labeled precursor 16 to zero by the use of C2-13 fragments. Presented here, are the relative portions of the ions M plus one to M plus three and M plus 16 to M plus 20 of the five most heavily labeled NLFA and PLFA from Protaphorura fimata, estimated on day zero and day one. While attempting this procedure, it is important to remember that consumers naturally comprise certain C isotopes.
This amount must be determined in control experiments without label diet. This natural certain C signal send subtracted as background from the label templates. Don't forget that working with solvent can be extremely hazardous, and precautions should be taken.
