Metabolic Pathway Confirmation and Discovery Through ¹³C-labeling of Proteinogenic Amino Acids

The goal of this protocol is to study bacterial metabolism using carbon 13 assisted metabolism analysis. To begin, microbes are grown in the presence of labeled carbon substrates, which are incorporated into the microbial proteins. The microbes are then harvested and amino acids are extracted, hydrolyzed and derivatized.

The samples are applied to a gas chromatograph and mass spectrometer or GCMS. By tracing the atom transition paths between metabolites in the biochemical network, the functional metabolic pathways utilized by the microbe can be determined. I'm Bert Burla.

The method we present here can be used as a compliment to transcriptomics and proteomics for determining the metabolic pathways in microbes. We have used this technique to investigate photosynthetic microbes such as cyanobacteria for myotrophic or heterotrophic carbon utilization. In this video, we will use ANOTHE 5 1 1 42 as a model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Demonstrating the procedure will be myself, li Yu and Chu Yang Fang. Begin this procedure by growing cyana thesei bacteria in a unlabeled medium. When the bacteria reach middle log growth phase, inoculate 3%by volume into the carbon 13 labeled medium.

Then to avoid the introduction of unlabeled carbon from the initial inoculum subculture into the same labeled medium at middle log growth phase. When the carbon 13 labeled Cy Thesei bacteria reach middle log growth phase, harvest 10 milliliters by centrifugation. Re suspend the pellet in 1.5 milliliters of six molar hydrochloric acid and transfer it to a clear glass screw top gas chromatography or GC vial.

Cap the vials and place them in a 100 degree Celsius oven for 24 hours to hydrolyze the biomass proteins into amino acids. Hydrolysis of biomass pellets yield 16 of the 20 common amino acids following hydrolysis, transfer the amino acid solution to two milliliter micro centrifuge tubes then centrifuge at 20, 000 Gs for five minutes. Following hydrolysis, transfer the amino acid solution to two milliliter micro centrifuge tubes then centrifuge at 20, 000 GS for five minutes to deposit solid particles.

After the spin, transfer the supernatants to new GC vials to remove solid particles in the hydrolysis solution, so they do not enter the GC column. Next, the amino acids are dert to render them. Volatile for separation by gas chromatography dissolve the dried samples with 150 microliters of tetrahedran, THF and 150 microliters of enter butyl dimethyl silo and methyl tri fluoro.

Acetamide, T-B-D-M-S Derivitization reagent incubate all of the samples in an oven between 65 and 80 degrees Celsius for one hour. Vortex the samples occasionally to make sure the metabolites in the vial are dissolved. Centrifuge the samples at 20, 000 GS for 10 minutes and then transfer the supernatant to new GC vials.

The supernatant should be a clear yellowish solution program. The GC temperatures as follows, hold at 150 degrees Celsius for two minutes. Increase at three degrees Celsius per minute to 280 degrees Celsius.

Increase at 20 degrees Celsius per minute to 300 degrees Celsius, and then hold for five minutes. Then for a 30 meter GC column, set the solvent delay to about five minutes and the range of the mass to charge ratio to between 60 and 500. Analyze the samples by GCMS using a one to five or one to 10 split ratio injection volume of one microliter and carrier gas helium of 1.2 milliliters per minute following the run.

Analyze the GCMS data using Microsoft Excel. The GC retention time and the unique mass to charge ratio peaks for each amino acid are illustrated in this figure.T-B-D-M-S. Deriv amino acids are usually clearly split by MS into two charged fragments fragment M minus 57, which contains the entire amino acid and fragment M minus 1 59, which lacks the alpha carboxyl group of the amino acid for leucine and iso leucine.

The M minus 57 peak was overlapped by other mass peaks fragment. N minus 15 can be used to analyze the entire amino acid labeling. Also, the F 3 0 2 group is often detected in most amino acids, and it contains only the first alpha carboxyl group and second carbons in an amino acid backbone.

However, because this Ms.Peak often has high noise to signal ratios, F 3 0 2 is not recommended for quantitatively analyzing the metabolic fluxes. Derivitization of amino acids or central metabolites introduces significant amounts of naturally labeled isotopes, including carbon 13, oxygen 18, silicon 29, and silicon 30. The measurement noise from natural isotopes in the raw mass isotope or spectrum can be corrected by using published software.

The final isotopic labeling data are reported as mass fractions. For example, M zero, M1, M two, M three, and M four, representing fragments containing zero to four carbon 13 labeled carbons cyana. These 5 1 1 2 does not contain the enzyme three anine ammonia lyase, which catalyzes conversion of three anine to two keto butyrate in the typical isle synthesis pathway.

Therefore, to resolve the isle pathway in this organism, cyana THESEI 5 1 1 42 was grown in ASP two medium with 54 millimolar glycerol and analyzed by GCMS as described in this video as shown here, Cyana Thesei 5 1 1 4 2 utilizes second position labeled glycerol as the main carbon source, and we observed that thianine and alanine whose precursor is pyruvate, have one labeled carbon while ISO leucine was labeled with three carbons. Therefore, synthesis and cyana, these 5 1 1 4 2 cannot be derived from the thianine route employed by most organisms. On the other hand, leucine and ISO leucine have identical labeling patterns based on fragment M minus 15 and fragment M minus 1 59.

For example, the isotope or data from N minus 15 containing UNFRAGMENTED amino acids showed identical labeling for leucine and ISO leucine. Thus leucine and ISO leucine must be synthesized from the same precursors, pyruvate and acetyl-CoA. This observation is consistent with the labeled carbon transition in the Citra malate pathway for isle synthesis.

This method can be used to investigate the metabolism of a wide diversity of microbes, and once mastered, can be performed from start to finish in about one week.