Extraction of Cofactor F₄₂₀ for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

The cofactor F420 plays an important role in the primary and secondary metabolism of many prokaryotes. Measuring this molecule in environmental samples enables a deeper understanding of its prevalence and function. This technique enables the analysis of the cofactor in difficult sample materials such sludge and soil.

Thereby the lysis of the material and pre-purification before analysis are the central steps. The protocol includes different steps during the sample preparation. These steps must be planned and prepared very well before starting with the sample processing.

For example, the preparation of fresh buffers is important. To begin the sample lysis, place five grams of the sample in a 50-milliliter conical tube. Then add five milliliters of 2X lysis buffer to the samples, and bring the volume to 10 milliliters with distilled water to reach a final concentration of 0.5 grams per milliliter.

Vortex the diluted samples for 20 seconds followed by autoclaving for 30 minutes at 121 degrees Celsius. For dry samples like forest soil, bring to a final volume of 20 milliliters with distilled water after autoclaving, and vortex the diluted sample again for 20 seconds as demonstrated. Once the samples cool down, centrifuge the sample lysate for five minutes at 11, 000 x g, and collect the supernatant.

Prepare a five-milliliter solid-phase extraction or SPE column filled with 100 milligrams of weak anion mixed-mode polymeric sorbent conditioning the anion exchanger with three milliliters of methanol. Then, equilibrate the anion exchanger with three milliliters of distilled water. Load up to nine milliliters of the supernatant from the centrifuged lysate onto the SPE column.

Sequentially wash the impurities from the column with five milliliters of 25-millimolar ammonia acetate and five milliliters of methanol. After washing, elute the cofactor F420 with one milliliter of elution buffer. Preset the oven to 40 degrees Celsius, and set the fluorescence detector to 420 nanometers extinction wavelength and 470 nanometers emission wavelength.

Next, filter the eluted sample from the SPE into HPLC vials using a PTFE filter with a pore size of 0.22 micron. Inject 50 microliters of the filtered sample into the HPLC system, and separate cofactor F420 via gradient mode using a combination of mobile phases as described in the manuscript. Analyze the cofactor F420 composition and concentration.

The growth of pure cultures of methanogens was verified by microscopy. In phase contrast microscopy, agglomerates of methanogens are visible, which emit a bluish light when cofactor F420 is excited with ultraviolet light. The peak area of recovered cofactor F420 after SPE with different volumes of the Methanoculleus thermophilus cultures was analyzed.

The autoclaving disintegration strategy achieved the highest peak area, concluding maximum extraction efficiency using a pressure-temperature treatment. Analysis of the tail length distribution revealed the differences in the overall concentration of cofactor F420 and the distribution of F420 tail length of the pure methanogenic cultures in environmental samples. When applying this procedure, be aware to completely collect total volume of the elution buffer or record the exact volume of flow-through.

This technique paves the way for the exploration of cofactor F420 in more complex samples like soils and sludges and enables deeper insights into the ecological impact of this molecule.