Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis

This protocol will aid users and teach students the correct steps in the extraction of BMA, AEG, and 2, 4-DAB. Thereby minimizing the chances of error and producing consistent results. This method is a commonly used and validated method for the extraction of BMA and its isomers, AEG and 2, 4-DAB in a sauna bacterial matrix.

This method can be utilized to further research regarding BMAs toxicity and provide a greater understanding about BMA and its associated health effects. Begin by centrifusion the cyanobacteria sample at 3, 500 G for 10 minutes at 25 degrees Celsius. And discarding the supernatant into biological waste.

Cover the centrifuge tube containing the pellet with a sealing film and pierce a few holes in the film using a sharp long nose tweezer. Then store the tube upright at minus 80 degrees Celsius. After 30 minutes, place the tube upright into a freeze dryer glass container and place the container inside the minus 80 degrees Celsius freezer for five minutes for cooling.

Remove the glass container from the freezer and attach the rubber lid. Ensure the handle on the rubber valve outlet of the freeze dryer is pointing upward and vented to the atmosphere. Attach the glass container firmly.

Slowly turn the handle of the rubber valve outlet to point it in a down position to expose the jar to the vacuum. And allow up to 24 hours of freeze drying to sublimate all the liquid. When done, weigh 15 to 50 milligrams of frozen sample pellets into a 15 milliliter centrifuge tube.

Using an analytical balance. To the sample, add 300 to 600 microliters of 10%aqueous Trichloroacetic acid or TCA. Place the sample tubes in a container filled with crushed ice.

Wipe the sonicator probe with a lint-free paper wipe sprayed with 70%ethanol before fully immersing the end of the probe into the sample and pressing start. Once done, place the sample containing the centrifuge tube on ice for one minute. And repeat the sonication process to ensure cell lysis.

After cooling the sample at four degree Celsius for 24 hours. Centrifuge at 3, 500 G for 15 minutes at eight degrees Celsius. Then using a micro pipette, transfer the supernatant to a two milliliter tube labeled free fraction.

Add 400 microliters of 10%aqueous TCA into the sample pellet, and break up the pellet either by vortex agitation or with the micro pipette tip. Centrifuge and transfer the supernatant into the same two milliliter free fraction tube. Repeat this step a third time using 10%TCA acetone.

Place the free fraction tube with the lid open into a centrifugal evaporator until all the volatile liquid is removed. Once the sample is free of volatile liquids, cover the tube securely with the ceiling film and pierce the film with a few holes using a sharp long nose tweezer. After freeze drying demonstrated before, reconstitute the sample with 200 microliters of 20 millimolar hydrochloric acid and place it in a minus 80 degree Celsius freezer.

Add 400 microliters of 100%acetone onto the sample pellet using a micro pipette and break up the pellet using either vortex agitation or the micro pipette tip. Quantitatively transfer the washed and resuspended pellet to the corresponding glass shell vial. Using a one milliliter micro pipette.

Add another 400 microliters of acetone to the sample tube to help transfer the remaining pellet. Centrifuge and decant the supernatant liquid into biological waste before drying the pellet in a centrifugal evaporator until the liquid is removed and the pellet is dry. To prepare the vacuum hydrolysis file add one milliliter of six molar hydrochloric acid to the bottom of the hydrolysis file.

Carefully insert the labeled shell vials with dried samples into the hydrolysis file using tweezers ensuring an upright stable position. Attach the lid to the hydrolysis file and push the red knob on the lid to close the valve. After turning on the vacuum pump, attach the vacuum tube to the head of the hydrolysis file lid and press the green knob on the lid to open the valve.

Next, allow the vacuum pump to remove the air from the vial for one minute. Then close the vial by depressing the red knob on the hydrolysis vial lid. Turn off the vacuum pump and remove the vacuum tube.

Attach a rubber tube to the nitrogen gas tap on the laboratory bench. And open the tap slightly. Place the thumb at the end of the tube, seal it and count until the gas begins to escape as pressure builds up.

Next, attach the other end of the rubber tube to the head of the hydrolysis vial lid. And immediately push the green knob of the lid. Count the time taken for the gas to escape.

Quickly push the red knob on the lid and remove the rubber tube. Repeat the entire vacuum treatment process twice. Ensuring the glass hydrolysis vial is free of air and filled with nitrogen gas.

Place the hydrolysis vial into a preheated oven set at 110 degrees Celsius for 16 to 18 hours. Using oven gloves, remove the glass hydrolysis vial from the oven. Allow the vial to cool inside the fume hood for 10 minutes before pushing the green knob while facing it away to release pressure and gas.

Reconstitute the hydrolyzed sample pellets with 200 microliters of 20 molar hydrochloric acid into the shell vial and resuspend the pellet. Centrifuge the shell vials containing the reconstituted sample pellets for two minutes at 5, 300 G and 25 degrees Celsius. Transfer the reconstituted free fraction samples and protein fraction into the two milliliter filter tubes containing 0.2 micron pore membrane filters and labeled as free fraction and protein fraction.

Place the tubes for centrifugation for 30 minutes at 5, 000 G and 25 degrees Celsius. The multiple reaction monitoring chromatogram of a standard containing beta-Methylamino-L-alanine or BMAA and its isomers indicated by the color coded lines is shown here. A positive detection for all three isomers, BMAA, DAB and AEG was observed in the free fraction of the Merismopedia species collected from Lake Liddel.

Whereas the bound fraction of the Microcystis flos-aquae species collected from Waka Waterworks illustrated a negative result for BMAA and its isomers. The resuspended pellet must be quantitatively transferred into the corresponding shell vial to allow an accurate point of normalization based on Cyanobacteria mass. Patients and due diligence are advised while performing this step.

Following this procedure. The extracted fractions will have to undergo a devitalization step to allow for reverse phase. Liquid chromatography, tandem mass spectrometry analysis.

The application of this technique expands various scientific fields. Including the environmental sciences through the analysis of cynotoxins in environmental samples. And fields such as toxicology and biochemistry through the investigation of the Misincorporation theory of BMA.