Systems-wide studies are crucial for the in-depth understanding of the biological functions. However, multiple independent samples are required for different omics platforms, introducing high amount of variability to the data. This method offers a robust and high-throughput strategy for simultaneous extraction of chlorophyll, lipids, metabolites, proteins, and starch from a single sample of the model green alga, Chlamydomonas reinhardtii.
For chlorophyll, lipid, and metabolite extraction, arrange the tubes of harvested Chlamydomonas cell pellets in liquid nitrogen. Resuspend the pellet in each tube with one milliliter of 20 degree extraction buffer one. To avoid evaporation of the low viscosity extraction buffer, quickly vortex the tubes until the cells are well homogenized within the extraction mixture and aliquot the solution into two milliliter microcentrifuge tubes.
Sonicate the cultures in a sonication bath in ice cold water for 10 minutes before incubation on an orbital shaker at 1, 000 rotations per minute for 60 minutes at four degrees Celsius. At the end of the incubation, add 650 microliters of extraction buffer two and briefly vortex the samples before centrifugation. To aliquot the fractions, transfer 500 microliters of the upper MTBE lipid phase into a labeled 1.5 milliliter tube.
Use a 200 microliter pipette to remove the lipid phase and transfer 650 microliters of the lower polar and semipolar metabolite phase into new labeled tubes. Aspirate the excess volume to remove the remaining lower phase and freeze the solid pellets in liquid nitrogen for 80 degree storage. For polar metabolite determination, resuspend the dried pellet of the polar phase in methoxyamine hydrochloride pyridine solution for methoxymization of the carbonyl groups and heat the samples at 30 degrees Celsius for 90 minutes.
At the end of the incubation, derivatize the samples with n-methyl-n-trifluoracetamide for 30 minutes at 37 degrees Celsius according to standard protocols. Use gas chromatography coupled to time-of-flight mass spectrometry to analyze the primary metabolites. For nonpolar metabolite determination, resuspend the dried pellet of the nonpolar phase in a mixture of seven to three volume to volume acetonitrile to isopropanol and sediment the pellets by centrifugation.
Then separate the samples on a reverse phase C8 column on an ultraperformance liquid chromatography system. For chlorophyll content determination, mix 100 microliters of the MTBE phase with 900 microliters of 90%methanol for method blank as well as the experimental samples. Then measure the absorbance on a spectrophotometer at 655 and 652 nanometer wavelengths to distinguish between chlorophyll a and chlorophyll b and calculate the chlorophyll a and b content and the total chlorophyll content.
For protein extraction, dissolve the lower phase pellet in 200 microliters of protein buffer and incubate the sample at room temperature for 30 minutes. At the end of the incubation, centrifuge the sample and transfer the protein-containing supernatant to a new tube. Measure protein concentration using the Bradford assay.
To digest the protein, reduce 15 micrograms of the sample in five millimolar dithiothreitol for 30 minutes, followed by alkylation with 10 millimolar iodoacetamide for 30 minutes at room temperature, protected from light. At the end of the alkylation, mix Trypsin/Lys-C solution at a 25 to one protein to protease ratio and incubate the sample for three hours at 37 degrees Celsius. Dilute the sample sixfold in 50 millimolar tris Hcl for an overnight incubation at 37 degrees Celsius.
The next morning, terminate the digestion with trifluoroacetic acid to a final concentration of 0.5 to 1%After the digestion, concentrate the samples to near dryness, leaving two to five microliters of solution in a vacuum concentrator without heating. Resuspend the sample in loading buffer. Analyze the peptide mixtures by liquid chromatography tandem mass spectrometry using a high resolution mass spectrometer connected to a nano ultra pressure liquid chromatography system.
To separate the peptides, load four microliters of sample onto a 20 centimeter reverse phase charged surface hybrid column with an inner diameter of 75 micrometers and a particle size of 1.7 micrometers. Use partial loop offline settings with an isocratic gradient set at 3%of buffer B, held for 14 minutes before the loop is shifted to the online position with the column, after which the gradient will be increased linearly for 50 minutes until 20%buffer B is reached. A shift in the cell volume can be observed as the cells grow in size throughout the light phase, followed by the release of daughter cells starting at the end of light phase from 10 hours.
Once all of the daughter cells have been released, a shift in the cell volume can be observed, as the newly released daughter cells are disposed to begin the next cycle. Based on the gas chromatography mass spectrometry analysis of the polar fraction, in this representative experiment, 65 metabolites were annotated. The liquid chromatography mass spectrometry analysis of the lipid-containing neutral phase led to the identification of 204 distinct lipid species covering various lipid classes.
Principal component analysis can be used to visualize global shifts in the metabolites and lipids across the cell cycle with a separation of light and dark phases as well as semi-cyclic phases observed for both metabolomics and lipidomic data. Here, an overview of the functional enrichment of 2, 463 identified enriched proteins is shown. The remaining pellet after protein extraction can be used for reproducible quantification of the starch, as indicated by the low standard deviation among various replicates.
It is important to avoid drying of the upper chlorophyll organic phase, as this can influence the levels of chlorophyll in the solvent, affecting the normalization factor for the samples. Various analytical procedures can be implemented for the biochemical characterization of the extracted molecular species.
