D.P.B., K.J.R., and S.M.M. are supported by The Ian Potter Foundation, and J.P.V. is the recipient of an Australian Government Research Training Program, Stipend.

The cyanobacteria species Merismopedia was used for the present study38.
1. Raw sample preparation
<ol>
	<li>Collect the algal scum from the aquatic source of interest or from a cyanobacterial cultured flask and place it into 50 mL centrifuge tube38. 
	NOTE: The sample must be frozen at &#8722;20 &#176;C for later extractions and thawed prior to the following step.</li>
	<li>Centrifuge the tubes containing the sample at 3,500 x g&#160;for 10 min at 25 &#730;C. Decant the supernatant into a waste container and discard it into biological waste. 
	NOTE: The supernatant may be reserved for future analysis of the exosome.</li>
	<li>Cover the tube containing the sample pellet securely with a sealing film (see Table of Materials) and pierce a few holes in the film using a sharp, long nose tweezer. Store the tube in an upright position at &#8722;80 &#176;C for 30 min.</li>
	<li>Turn on and equilibrate the freeze dryer at 0.1 mbar and &#8722;80 &#176;C (~30 min). 
	&#8203;NOTE: The parameters (step 1.4) are optimized for the freeze dryer used for the present study (see Table of Materials). Follow standard operating procedures according to the freeze dryer model available in the laboratory or set the lowest possible temperature if the freeze dryer model does not go as low as &#8722;80 &#730;C.
	<ol>
		<li>Place the centrifuge tube(s) upright into a freeze dryer glass container and place it inside the &#8722;80 &#176;C freezer for 5 min to cool the jar.</li>
		<li>Remove the glass container from the freezer and attach the rubber lid.</li>
		<li>Ensure the handle on the rubber valve outlet of the freeze dryer is vented to the atmosphere (pointing upward) and attach the glass container firmly.</li>
		<li>Turn the handle of the rubber valve outlet very slowly to the pointing down position to expose the jar to the vacuum and allow up to 24 h of freeze-drying to ensure the sublimation of all the liquid.</li>
		<li>To release the vacuum in the jar, turn the handle to the pointing upward position, detach the glass container, and remove the freeze-dried samples.</li>
	</ol>
	</li>
	<li>Use an analytical balance to weigh 15-50 mg of dried sample pellets into a 15 mL centrifuge tube. 
	&#8203;NOTE: The samples may be placed into &#8722;80 &#176;C storage if not processed further at this stage.</li>
</ol>
2. Cell lysing and fractionation of free NPAAs
<ol>
	<li>Add 100 &#181;L of 100 ng/mL&#160;of D5-2,4-DAB (see Table of Materials) standard using a micropipette to the sample tube (optional).</li>
	<li>Add 300-600 &#181;L of 10% w/v aqueous trichloroacetic (TCA, see Table of Materials) or 300-600 &#181;L of 11.7%-13.3% TCA, respectively, if adding the D5-2,4-DAB standard. 
	NOTE: Choose a volume of aqueous TCA that fully covers the pellet.</li>
	<li>Place the sample tubes in a container filled with crushed ice.</li>
	<li>Use a probe sonicator (see Table of Materials) at medium-high power (70%) and lyse the sample for 1 min following the steps below. 
	&#8203;CAUTION: Ensure proper PPE with the use of noise-canceling earmuffs.
	<ol>
		<li>Follow the appropriate safety protocols in preparation for the use of the probe sonicator by placing in-use signage on the door, performing sonication in the fume hood, and using noise-canceling earmuffs.</li>
		<li>Switch on the sonicator and enter the following parameters: amplitude, 70%; time, 1 min.</li>
		<li>Spray 70% ethanol onto a lint-free paper wipe (see Table of Materials) and wipe down the probe.</li>
		<li>Fully immerse the end of the probe into the sample, and press start.</li>
		<li>When the probe sonicator stops, place the centrifuge tube containing the sample on ice for 1 min.</li>
		<li>To ensure the cells are lysed, repeat steps 2.4.3-2.4.5 once more.</li>
	</ol>
	</li>
	<li>Place the sample into a fridge at 4 &#176;C for 12-24 h to allow protein precipitation.</li>
	<li>Centrifuge the 10% aqueous TCA sample at 3,500 x g for 15 min at 8 &#176;C.</li>
	<li>Use a micropipette to transfer the supernatant to a 2 mL tube labeled &#34;Free Fraction&#34;.</li>
	<li>Micropipette 400 &#181;L of 10% aqueous TCA into the centrifuge tube containing the remaining sample pellet and break up the pellet either by vortex agitation or with the micropipette tip.</li>
	<li>Repeat steps 2.6-2.7, transferring the supernatant to the same 2 mL &#34;Free Fraction&#34; tube.</li>
	<li>Micropipette 400 &#181;L of 10% TCA/acetone into the centrifuge tube with the remaining pellet and break up the pellet with vortexing or the micropipette tip.</li>
	<li>Centrifuge the 10% TCA/acetone sample at 3,500 x g&#160;for 15 min at 8 &#176;C. Use a micropipette to transfer the supernatant to the 2 mL tube labeled &#34;Free Fraction&#34;.</li>
	<li>Place the &#34;Free Fraction&#34; tube with the lid open into a centrifugal evaporator (see Table of Materials) until all the volatile liquids are removed (at least 1 h).</li>
	<li>Once the sample is free of volatile liquids, cover the tube securely with sealing film and pierce the film with a few holes using a sharp, long nose tweezer. Place the sample into a &#8722;80 &#176;C freezer.</li>
	<li>Freeze dry the &#34;Free Fraction&#34; sample by repeating all the steps in step 1.4.</li>
	<li>Micropipette 200 &#181;L of 20 mM hydrochloric acid (HCl) into the &#34;Free Fraction&#34; tube to reconstitute the freeze-dried sample and place it in &#8722;80 &#176;C freezer storage. 
	NOTE: The &#34;Free Fraction&#34; of the sample is ready for filtration in step 4. The remaining pellet will be further processed in the following steps for protein fractionation.</li>
</ol>
3. Fractionation of the protein-bound NPAAs
<ol>
	<li>Use a glass engraver to label glass shell vials with the sample details for identification. 
	NOTE: Strong acid may dissipate ink labeling; therefore, engraving the labels on glass is recommended.</li>
	<li>Micropipette 100 &#181;L of 100 ng/mL&#160;D5-2,4-DAB standard onto the sample pellet (optional).</li>
	<li>Micropipette 400 &#181;L of 100% acetone onto the sample pellet and break up the pellet using either vortex agitation or the micropipette tip.</li>
	<li>Use a 1 mL micropipette set at 1,000 &#181;L to transfer the washed and resuspended pellet to the corresponding glass shell vial. 
	NOTE: Another 400 &#181;L of 100% acetone can be added to the agitated pellet to assist in the complete transfer of the pellet into the glass shell vial. This can be repeated a third time if necessary.</li>
	<li>Centrifuge at 8,000 x g for 5 min at 25 &#730;C and decant the liquid into biological waste.</li>
	<li>Place the remaining pellet into a centrifugal evaporator until all the liquid is removed and the pellet is dry (~1 h).</li>
	<li>Prepare the vacuum hydrolysis vial by adding 1 mL of 6 M HCl into the bottom of the hydrolysis vial.</li>
	<li>Use tweezers to carefully insert the labeled shell vials containing the dried samples into the hydrolysis vial, ensuring an upright, stable position. 
	NOTE: Empty shell vials can be used to help keep the sample vials upright if the number of samples is less than the capacity of the hydrolysis vial.</li>
	<li>Attach the lid to the hydrolysis vial and push the red knob on the lid to close the valve.</li>
	<li>Turn the vacuum pump on, attach the vacuum tube to the head of the hydrolysis vial lid, and press the green knob on the lid to open the valve.</li>
	<li>Allow the vacuum pump (see Table of Materials) to remove the air from the vial for 1 min.</li>
	<li>Close the vial by depressing the red knob on the hydrolysis vial lid, turn the vacuum pump off, and remove the vacuum tube.</li>
	<li>Attach a rubber tube to the nitrogen gas tap on the laboratory bench and open the tap slightly. Place the thumb at the tube&#39;s end, seal it, and count until the gas begins to escape as pressure builds up. This will be the timeframe for the next step. Adjust the gas flow to a suitable timeframe.</li>
	<li>Attach the other end of the rubber tube to the head of the hydrolysis vial lid and then immediately push the green knob of the lid. Count to the time determined in step 3.13 and quickly push the red knob on the lid and remove the rubber tube.</li>
	<li>Repeat steps 3.10-3.14 twice to ensure the glass hydrolysis vial is free of air and filled with nitrogen gas.</li>
	<li>Place the hydrolysis vial into a preheated oven set at 110 &#176;C for 16-18 h.</li>
	<li>Use oven gloves to remove the glass hydrolysis vial from the oven and allow it to cool inside the fume hood for 10 min. Inside the fume hood, while facing it away from you, push the green knob to release pressure and gas.</li>
	<li>Use tweezers to remove the shell vials from the hydrolysis vial.</li>
	<li>Reconstitute the hydrolyzed sample pellets by micro pipetting 200 &#181;L of 20 mM HCl into the shell vial. Ensure the pellet is resuspended by either vortexing or using a pipette tip.</li>
	<li>Centrifuge the shell vials containing the reconstituted sample pellets for 2 min at 5,300 x g&#160;and 25 &#730;C.</li>
</ol>
4. Sample filtering
<ol>
	<li>Label 2 mL filter tubes (see Table of Materials) containing 0.2 &#181;m pore membrane filters as &#34;Free Fraction&#34; and &#34;Protein Fraction&#34;.</li>
	<li>Transfer the reconstituted samples from step 2.15 (free fraction) and step 3.20 (protein fraction) into the corresponding filter tubes.</li>
	<li>Place them in the centrifuge for 30 min at 5,000 x g&#160;and 25 &#730;C.</li>
	<li>Remove the filters from the filter tubes and cap them. The samples are now ready for amino acid derivatization in step 5. 
	&#8203;NOTE: The samples can be placed into the &#8722;80 &#176;C freezer for storage for later derivatization and analysis.</li>
</ol>
5. Amino acid derivatization
<ol>
	<li>Derivatize the samples using propyl chloroformate (PCF)39 or 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC)40 according to the manufacturer&#39;s instructions (see Table of Materials).</li>
</ol>

The authors have nothing to disclose.

The extraction protocol outlined here for the analysis of NPAAs applies to analyzing any amino acids in biological samples. For guides on isolating and culturing cyanobacterial strains, one can refer to the methods presented in the study by Violi et al.38. The first step in the protocol takes the sample to a point where normalization between samples against the dry weight can be achieved. The second step is cell lysis to release the analytes and can be performed using an array of techniques, including mechanical disruptions/lyses such as probe sonication as described in this protocol, freeze/thaw cycles, grinding and bead milling, and non-mechanical disruptions such as enzymatic, detergent and/or chemical lysis. Mechanical disruption is known to be advantageous over non-mechanical as it allows for a greater capacity of the sample to lyse while allowing the intracellular bonds and proteins to remain intact41, although the sample matrix may dictate the optimal method for cell lysing.
Protein precipitation is the critical third step of this protocol when extracting amino acids for analysis. TCA is the most commonly used solvent; however, perchloric acid, acetone, methyl tert-butyl ether (MTBE), methanol, and/or acetonitrile8,42 have also been used, where each extraction solvent is purposed to extract and precipitate different substrates. The model described here extracts the NPAA BMAA and its isomers from a cyanobacterial matrix, and although a range of different solvents have been utilized, the two most common are 10% TCA in water (aqueous) and 10% TCA in acetone. In general, protein precipitation with TCA is commonly used to extract amino acids to fractionate free amino acids from the protein-bound amino acids. Additionally, TCA extraction allows for determining the total protein content, reducing contaminants for the free fraction, and reducing the activity of proteases with minimal protein degradation43. The TCA extraction of the free fraction works by initially rinsing out organic-soluble substances, leaving behind proteins and insoluble compounds such as cell wall remnants in the precipitate, which is then followed by thermal hydrolysis extraction of the protein-bound amino acids (bound fraction) using a strong acid.
The fractionation of the free fraction with 10% aqueous TCA plus sonication produces the most extensive protein precipitation compared to other organic solvents44 and the best amino acid recoveries42. Some studies opt to use an acid combined with an organic solvent (i.e., 10%-20% TCA in acetone43) to precipitate more molecules, including smaller peptides/biomolecules, minimize protein degradation, and reduce contaminants such as salts43. Another advantage of 10% TCA in acetone is its faster drying rate in the preparation of the pellet for hydrolysis, minimizing moisture residue to prevent amino acid modification. However, 10% aqueous TCA plus sonication has better extraction efficiencies of free amino acids when compared to 10% TCA in acetone44 alone. Additionally, 10% aqueous TCA precipitation has limitations such as a long drying time, not being able to precipitate all proteins or small peptides/biomolecules, and depending on the analyte of interest (i.e., proteins or amino acids), requiring additives and reducing agents to prevent oxidization and degradation45,46.
This protocol uses a combination of 10% aqueous TCA and 10% TCA in acetone to increase protein precipitation, ensure smaller peptides/biomolecules are precipitated, allow for faster pellet drying times, and increase extraction efficiencies, taking advantage of both solvent extraction properties. However, the combination of 10% aqueous TCA and 10% TCA in acetone can precipitate small peptides/biomolecules in the free fraction after the 10% TCA in acetone supernatant is combined with the aqueous free fraction. In this case, the precipitates should be transferred to the bound fraction for hydrolysis.
The second half of this protocol involves the release of amino acids (i.e., BMAA) from the protein pellet via acid-vapor hydrolysis at elevated temperatures in an oxygen-free environment. The predominately limiting factor to the hydrolysis step is that it is time-consuming and laborious to prepare. The overnight incubation prevents fast and time-efficient sample preparation and analysis. In addition, the quantitative transfer of the pellet from a centrifuge tube to a shell vial (step 3.4) is an arduous process that requires diligence and patience to ensure an accurate point of normalization to the dry weight. Possible issues the user may face are the incomplete transfer of the pellet, wet precipitated proteins sticking to pipette tips and the original tube, and solid particles of the pellet blocking the pipette tip. A practical suggestion in aiding an easier transfer of the pellet is to remove about 0.3-0.5 mm off the end of the pipette tip with a pair of scissors, allowing larger pellet particles to be drawn and released into the shell vial. This transfer method is common practice for protein extraction for all amino acid analyses. However, modifications to improve the efficiency and accuracy of quantitatively transferring the pellet into the shell vial should be investigated.
Two forms of hydrolysis techniques can be employed to release amino acids from their protein-bound state: liquid-phase hydrolysis and acid-vapor hydrolysis. Liquid-phase hydrolysis, which involves the addition of 6 M HCl onto the sample, which is then placed in a 110 &#176;C oven overnight, is an alternative to the acid-vapor hydrolysis described in this protocol. Extraction techniques that employ liquid-phase hydrolysis may require further processing, such as desalination, especially if AQC derivatization is chosen, as it requires basic conditions for tagging40. Acid-vapor hydrolysis avoids desalination and allows the user the freedom to choose the derivatization technique upon reconstitution of the final pellet before filtration. Furthermore, independent of the chosen hydrolysis method, the samples may require further processing, such as SPE for matrix purification and concentration29,47,48, depending on the choice of derivatization technique and/or analytical analysis (e.g., reverse phase LC-MS/MS vs. hydrophilic interaction liquid chromatography [HILIC] LC-MS/MS37). The reconstitution of the final pellet in 20 mM HCl in this protocol is appropriate for derivatization using PCF reagents48&#160;via a commercially available amino acid hydrolysis kit39 (see Table of Materials). Both AQC and PCF will derivatize all the amino acids, protein, and non-protein in origin present in the sample, and the selectivity of the analytes is obtained through the use of LC-MS/MS and its combination of retention time matching and careful selection of the quantifier and qualifier MRMs38.
Current hydrolysis protocols are not optimized for the release of BMAA, 2,4-DAB, and AEG but for the 22 protein amino acids based on existing methods for protein hydrolysis32, where incubation for longer than 18 h results in the degradation and modification of certain amino acids, affecting LC-MS/MS analysis and, thus, the obtained concentrations32. Beach et al.49 investigated hydrolysis over time (0.5-120 h) to optimize hydrolysis for analyzing BMAA and the proteinogenic amino acids. They found that although there was an early rapid release of BMAA during the first 0.5 h of the hydrolysis, the BMAA levels continued to increase as the hydrolysis time increased, without degradation, even after 5 days49. There are also still different views and questions about the true nature of bound BMAA and its isomers, with some studies suggesting that rather than BMAA bonding50,51 or misincorporation into proteins14, BMAA-protein association is superficial52. However, the current consensus in the analysis of &#34;bound&#34; BMAA requires the validated and widely accepted hydrolysis step that breaks apart peptides/proteins to release amino acids and, thus, BMAA and its isomers.
The recovery rates of 20 protein amino acids were determined in a study by Sedgwick et al.42 using TCA extraction, resulting in approximately 100% recovery. In the case of BMAA and its isomers from algal matrices, studies comparing the efficiency of BMAA extraction using various extraction solvents have found 10% TCA to be the most efficient for free BMAA27. The recovery rates for protein-bound BMAA extracted using liquid-phase hydrolysis were established in studies by Glover et al.25 and by Faassen et al.53, where the accuracy was determined by spiked recovery methods, with an average BMAA recovery rate of 108.6% and 70%, respectively. Faassen et al. described procedures for spike-recovery experiments and illustrated how the recovery differs depending on the stage of the extraction process the spike was made53. Faassen et al. additionally determined the efficiency of the acid-vapor protocol presented here, with recovery rates of 83.6% for the BMAA isomers spiked prior to extraction and 68.6% for those spiked prior to hydrolysis24. These recoveries, methods of extraction, and analyses were further validated by Banack26, with recovery rates of 94%-106% for BMAA in a cyanobacterial matrix and a %Relative Standard Deviation (%RSD) between 5.6% and 20%, meeting the FDA criteria for accuracy54. It is recommended that each laboratory initially establish its recovery rates and accuracy before utilizing this protocol via spike recovery experiments. The recovery methods presented in Faassen et al.53 and Glover et al.25 may be followed as a guide.
Alternative forms of hydrolysis techniques can be utilized instead of overnight oven hydrolysis for faster protein-bound extraction of the analyte. For example, a microwave extractor could significantly reduce the hydrolysis time from 24 h to as little as 10 min55. Microwave hydrolysis for amino acids uses the same principles as the conventional thermal method, using a glove box to prepare and assemble the microwave vessel in an oxygen-free environment and adding 6 M HCl. Once air-tight, the vessel containing the sample undergoes microwave radiation. Microwave hydrolysis has been used to extract amino acids from various sample matrices56,57,58; however, microwave hydrolysis has yet to be developed and validated for the analysis of BMAA.
In conclusion, 10% aqueous TCA protein precipitation is the optimal method to extract free non-protein amino acids from cyanobacterial matrices27, and thermal hydrolysis provides a reliable and reproducible extraction of the protein-bound fraction. This protocol to extract the NPAA BMAA and its isomers from cyanobacteria is validated and widely accepted. Future directions to further optimize the extraction of BMAA will focus on the hydrolysis step, which is performed according to the specifications of the 22 protein amino acids. However, the extraction protocol presented here should still be considered efficient and effective for analyzing BMAA and its isomers via LC-MS/MS.

Amino acids are chemical compounds that contain at least one amine and carboxylic functional group. Some amino acids also contain an imino group, a functional acid group other than carboxylic; other amino acids have amine groups that are not attached to the &#945;-carbon group1. There are over 500 amino acids2, 22 of which are known as protein amino acids used for genetic coding in ribosomal protein synthesis3. These 22 amino acids can be further subdivided into essential and non-essential. Essential amino acids are necessary for an organism to function properly and can only be acquired by external sources. Non-essential amino acids can be synthesized within the organism. The classification of the 22 amino acids into essential/non-essential is unique to individual species. All other amino acids are non-protein amino acids (NPAAs) that are not encoded for protein synthesis. Amino acids, whether protein or non-protein, can also play a signaling role within an organism and act as metabolic intermediaries4. Due to their important and varying roles, amino acid levels can provide an insight into organism condition, functionality, and metabolic pathways, etc. There are two main mechanisms by which amino acids can be incorporated into a polypeptide chain: ribosomal protein synthesis, which utilizes the 22 amino acids in encoding, and non-ribosomal peptide synthesis, which, alongside protein amino acids, allows for the use of some NPAAs in synthesis. Certain NPAAs can mimic protein amino acids, potentially leading to their misincorporation into peptides and proteins. The misincorporation causes a misfolding of proteins, which, in turn, has detrimental effects5, such as the misincorporation of the NPAA L-3,4 dihydroxyphenylalanine (L-DOPA) in place of tyrosine, negatively impacting cellular function and health6,7. An additional source of incorporated NPAAs is through the post-translational modification (PTM) of amino acid residues. Amino acid residues are modified for various reasons, including changes to peptide or protein conformation, stability, and functionality. Upon hydrolysis of PTM-containing protein or peptides, these modified amino acid residues are released into their free NPAA form8,9.
The NPAA &#946;-methylamino-L-alanine (BMAA) produced by cyanobacteria, diatoms, and dinoflagellates10 is a suspected neurotoxin that is implicated as a contributing factor to various neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC)11,12, amyotrophic lateral sclerosis, and Alzheimer&#39;s disease13. It is suggested that BMAA is mistakenly incorporated into the polypeptide chain of proteins in place of L-serine14 and/or other protein amino acids. The misincorporation of BMAA can lead to the misfolding of proteins, resulting in the deposition of protein aggregates in neurons14. In the last decade, interest in BMAA has significantly increased. A wide range of cyanobacterial species from freshwater, marine, and brackish environments have been discovered to produce BMAA15, leading to their widespread distribution into various ecosystems16,17. Additionally, BMAA has been shown to biomagnify through the food chain to the human food web18,19. Due to the potential health effects, lack of understanding, and incongruences of BMAA toxicity, it is imperative to continue further research until either the toxicity of BMAA is ultimately understood or BMAA is deemed safe20,21.
The analysis of amino acids in biological samples can be divided into four main steps: sample preparation, amino acid derivatization, separation and detection, and identification and quantification. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the preferred analysis method as it provides a targeted and reproducible separation and analysis of amino acids.
Sample preparation techniques for analyzing cyanobacterial and other algal samples predominantly involve an extraction method for amino acids in their free and protein-bound forms. Over the years, the extraction methods have remained relatively consistent with common elements, including the desolvation of the sample to separate the free amino acids from their bound form, followed by protein precipitation and the release of the bound amino acids through hydrolysis with hydrochloric acid (HCl) at elevated temperatures22. This extraction form has been optimized for protein amino acids and employed for NPAAs. However, the detection and quantification of BMAA and its isomers (Figure 1), aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB), in the same species of cyanobacteria have shown inconsistent results in the literature, with a possible explanation lying in differences in the growth conditions and/or the strain of algae producing varying or no amounts of BMAA23. It has been argued that a more likely explanation for the inconsistencies in the detection and quantification of BMAA and its isomers is due to unvalidated experimental protocols, the use of a wide range of analytical techniques, and insufficient experimental detail in the reported methods16,24 leading to irreproducible inter-laboratory data. However, Glover et al.25 and Banack26 have recently developed and validated an analytical technique for the detection and quantification of BMAA and its isomers using ultra-performance liquid chromatography (UPLC)-MS/MS in accordance with the International Society of Analytical Chemists (AOAC), US Pharmacopeia, and FDA guidelines necessary for single-laboratory validation.
These validation experiments focused on the separation and detection of BMAA and its isomers and did not address the inconsistencies in sample preparation protocols. Lage et al.27 compared the performance of three common extraction methods for quantifying BMAA and its isomers in cyanobacterial samples via LC-MS/MS: solid phase extraction (SPE) of free amino acids28,29; a protein precipitation method involving a methanol extraction and acetone precipitation30; and the most commonly used extraction method for BMAA, protein precipitation with trichloroacetic acid (TCA)31. They concluded that the TCA protein precipitation was the optimal protocol, yielding higher BMAA concentrations in the test samples compared to the other extraction methods. Their study validated TCA extraction with derivatization of BMAA using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in a cyanobacteria matrix, providing an established guide to achieving reliable and reproducible BMAA data. TCA amino acid extraction is an accepted and common sample preparation technique that can also be applied to other matrices. However, the stability of the amino acid during hydrolysis needs to be considered to prevent degradation or oxidation, which can be overcome with the use of chemical modifiers and reducing agents32. TCA extraction is routinely used and taught to new research students, and although the protocol is widely reported, a visual aid in the application of this method is a valuable resource, ensuring proper and consistent execution.
Reverse-phase chromatography is commonly used to separate amino acids, requiring a derivatization step prior to analyses. The derivatization of amino acids such as BMAA allows for chromatographic retention and can increase resolution between isomers. It also increases the molecular mass and improves ionization in the mass spectrometer. Several derivatizing reagents have been used for the analysis of amino acids via LC-MS/MS, including propyl chloroformate (PCF)33, 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC)27, 9-fluorenylmethyl chloroformate (FMOC)34, and dansyl chloride (DC)35. However, the only validated techniques for analyzing BMAA used either PCF36 or AQC24,26,37 as their derivatizing reagent.
The scope of this protocol is focused on the TCA extraction of NPAAs from cyanobacterial matrices. It is a labor-intensive method that is routinely used and taught in academic and industry research laboratories based on manuscripts that may be short on detail; therefore, this protocol provides details of the procedure and techniques involved in preparing samples for the analysis of free and bound BMAA as the model amino acid.

<table><tbody><tr><td>1 mL glass shell vials</td><td>SHIMADZU</td><td>REST-24663</td><td></td></tr><tr><td>10% TCA solution</td><td></td><td></td><td>Dilute 100% (w/v) to 10% aqueous TCA&amp;nbsp;</td></tr><tr><td>100% TCA solution</td><td></td><td></td><td>Dissolve TCA in water according to the following ratio 2.2 g:1 mL (TCA:H&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>1000 &amp;micro;L micropipette</td><td>Ependorf/Sigma-Aldrich</td><td>Z683809</td><td></td></tr><tr><td>13.3% TCA solution</td><td></td><td></td><td>Dilute 100% (w/v) to 13.3% aqueous TCA&amp;nbsp;</td></tr><tr><td>2 mL tubes</td><td>MERCK</td><td>BR780546-500EA</td><td></td></tr><tr><td>20 mM HCl</td><td>Chem-Supply Pty Ltd</td><td>7647-01-0</td><td>Made from stock</td></tr><tr><td>20-200 &amp;micro;L micropipette</td><td>Ependorf/Sigma-Aldrich</td><td>Z740441</td><td></td></tr><tr><td>6 M HCl</td><td>Chem-Supply Pty Ltd</td><td>7647-01-0</td><td>Made from stock</td></tr><tr><td>70% ethanol</td><td>Supelco</td><td>1.11727</td><td>Dilute 70:1 Ethanol:water</td></tr><tr><td>-80 &amp;deg;C Freezer</td><td>Martin CHRIST</td><td>102142</td><td>Alpha 2-4 Ldplus</td></tr><tr><td>AccQ-Tag Ultra Derivatisation Kit&amp;nbsp;</td><td>Waters</td><td>186003836</td><td></td></tr><tr><td>Acetone</td><td>Chem-Supply Pty Ltd</td><td>34967-2.5L</td><td></td></tr><tr><td>Analytical Scale Balance</td><td></td><td></td><td></td></tr><tr><td>Centrifugal evaporator</td><td>Thermo Scientific</td><td>13442549</td><td>DNA120-115 SpeedVac Concentrator</td></tr><tr><td>Centrifuge</td><td>MERCK</td><td>EP022620100-1EA</td><td></td></tr><tr><td>D5-2,4-DAB standard</td><td>CDN Isotopes</td><td>D-7568</td><td></td></tr><tr><td>Drying Oven</td><td></td><td></td><td></td></tr><tr><td>Ez:faastTM Amino Acid Analysis Kit</td><td>Phenomenex</td><td>CEO-8492</td><td></td></tr><tr><td>Falcon tube holder</td><td></td><td></td><td></td></tr><tr><td>Falcon Tubes</td><td>MERCK</td><td>T2318-500EA</td><td>Greiner centrifuge tubes</td></tr><tr><td>Filter Tubes</td><td>Sigma-Aldrich</td><td>CLS8161-100EA</td><td>Corning Costar Spin-X centrifuge tube filters (pore size 0.22 &amp;mu;m)</td></tr><tr><td>Glass engraver</td><td></td><td></td><td></td></tr><tr><td>Hydrolysis vial &amp;amp; Lid</td><td>Eldex Laboratories/Waters</td><td>WAT007568 &amp;amp; WAT007569</td><td></td></tr><tr><td>Ice and container</td><td></td><td></td><td></td></tr><tr><td>Lint-free paper wipe</td><td>Kimwipes/Sigma-Aldrich</td><td>Z188956</td><td></td></tr><tr><td>Milli-Q water</td><td></td><td></td><td>18.2 M&amp;Omega;.cm</td></tr><tr><td>Nitrogen tap with hose</td><td></td><td></td><td></td></tr><tr><td>P1000 Micropipette</td><td>MERCK</td><td>EP3123000063</td><td></td></tr><tr><td>P1000 pipette tips</td><td>MERCK</td><td>Z740127</td><td></td></tr><tr><td>P200 Micropipette</td><td>MERCK</td><td>EP3123000055-1EA</td><td></td></tr><tr><td>P200 pipette tips</td><td>MERCK</td><td>Z740125</td><td></td></tr><tr><td>Parafilm</td><td>MERCK</td><td>P7793</td><td>Sealing film</td></tr><tr><td>PPE - noise cancelling headphones</td><td></td><td></td><td></td></tr><tr><td>PPE - oven gloves</td><td></td><td></td><td></td></tr><tr><td>Probe sonicator</td><td>QSONICA Sonicators</td><td>Q125 Sonicator</td><td>Please ensure all appropriate PPE (i.e. noise cancelling headphones)</td></tr><tr><td>Sample transfer spatula</td><td></td><td></td><td></td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Sigma-Aldrich</td><td>T6399-1KG</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Vacuum Pump with hose</td><td></td><td></td><td></td></tr></tbody></table>

extraction of non-protein amino acids from cyanobacteria for liquid chromatography-tandem mass spectrometry analysis

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of NPAAs can provide crucial information about cellular uptake and/or function, metabolic pathways, and potential toxicity. &#946;-methylamino-L-alanine (BMAA) is a neurotoxic NPAA produced by various algae species and is associated with an increased risk for neurodegenerative diseases, which has led to significant research interest. There are numerous ways to extract AAs for analysis, with liquid chromatography-tandem mass spectrometry being the most common, requiring protein precipitation followed by acid hydrolysis of the protein pellet. Studies on the presence of BMAA in algal species provide contradictory results, with the use of unvalidated sample preparation/extraction and analysis a primary cause. Like most NPAAs, protein precipitation in 10% aqueous TCA and hydrolysis with fuming HCl is the most appropriate form of extraction for BMAA and its isomers aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB). The present protocol describes the steps in a validated NPAA extraction method commonly used in research and teaching laboratories.

An illustration of the extraction protocol is provided in Figure 2 as a summarized reference guide. The results obtained by Violi et al.38 were chosen to represent a positive result from this extraction protocol for the analysis of BMAA isomers from cyanobacteria. Nineteen single species of cyanobacteria were cultured from 11 eastern Australian freshwater sites. Using the same protocol, the BMAA isomers were extracted into free and protein fractions, derivatized with PCF, and analyzed using LC-MS/MS. Seventeen of the cyanobacterial isolates were positive for BMAA, and all 19 contained the 2,4-DAB isomer. A positive result is confirmed by using a validated LC-MS/MS method and observing at least three multiple reaction monitoring (MRM) transitions, one as quantifier ion and two as qualifier ions at the expected retention time. A representative MRM chromatogram of a standard containing BMAA and its isomers, indicated by the color-coded lines, is shown in Figure 3. The presence and concentration of BMAA and isomers in all 19 samples, sectioned to show the BMAA and isomer concentration in the free and bound fractions, is summarized in Table 1. A positive detection for all three isomers was observed in the free fraction of the Merismopedia species collected from Lake Liddell (NSW, Australia), where the free fraction contained a BMAA concentration of 68.38 &#181;g/g dry weight (DW) &#177; 2.25 &#181;g/g DW, 2,4-DAB with a concentration of 1,223.98 &#181;g/g DW &#177; 20.7 &#181;g/g DW, and AEG with a concentration of 125.27 &#181;g/g DW &#177; 4.19 &#181;g/g DW. The chromatographic MRMs are shown in Figure 4. To illustrate a negative result for BMAA and its isomers in a sample, the bound fraction of the Microcystis flos-aquae species collected from Walka Water Works (NSW, Australia) is chromatographically presented in Figure 5. While the bound fraction contained no BMAA, 2,4-DAB, and/or AEG, its free fraction contained all three isomers, with concentrations of 79.86 &#181;g/g DW &#177; 1.59 &#181;g/g DW, 1,156.15 &#181;g/g DW &#177; 8.46 &#181;g/g DW, and 433.83 &#181;g/g DW &#177; 8.92 &#181;g/g DW, respectively.
Thus, through the use of this protocol, Violi et al.38 confirmed the presence of BMAA isomers in eastern Australian freshwater cyanobacteria and determined which cyanobacteria had toxin-producing capabilities.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63779/63779fig01.jpg" /> 
Figure 1: The chemical structure of BMAA and its isomers 2,4-DAB and AEG. <a href="https://www.jove.com/files/ftp_upload/63779/63779fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63779/63779fig02.jpg" /> 
Figure 2: Summarized reference diagram of the extraction protocol. <a href="https://www.jove.com/files/ftp_upload/63779/63779fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63779/63779fig03.jpg" /> 
Figure 3: LC-MS/MS chromatogram of a calibration standard containing D5-2,4-DAB, 2,4-DAB, BMAA, and AEG for isomer identification based on retention, indicated by the highlighted peaks. Key: D5-2,4-DAB 338.01 m/z &#62; 278.10 m/z at 6.4 min (red), 2,4-DAB 333.01 m/z &#62; 273.10 m/z at 6.4 min (green), AEG 333.01 m/z &#62; 88.00 m/z at 7.4 min (orange), and BMAA 333.01 m/z &#62; 187.10 m/z at 7.8 min (blue). <a href="https://www.jove.com/files/ftp_upload/63779/63779fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63779/63779fig04.jpg" /> 
Figure 4: LC-MS/MS chromatogram for detecting D5-2,4-DAB, 2,4-DAB, BMAA, and AEG&#160;in Merismopedia species collected from Lake Liddell (NSW, Australia), indicated by the highlighted peaks. Key: D5-2,4-DAB 338.01 m/z &#62; 278.10 m/z at 6.4 min (red),&#160;2,4-DAB 333.01 m/z &#62; 273.10 m/z at 6.4 min (green), AEG 333.01 m/z &#62; 88.00 m/z at 7.4 min (orange), and BMAA 333.01 m/z &#62; 187.10 m/z at 7.8 min (blue). <a href="https://www.jove.com/files/ftp_upload/63779/63779fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63779/63779fig05.jpg" /> 
Figure 5: LC-MS/MS chromatogram of a negative control for 2,4-DAB, BMAA, and AEG in a bound fraction of Microcystis flos-aquae species collected from Walka Water Works (NSW, Australia). Key: D5-2,4-DAB 338.01 m/z &#62; 278.10 m/z at 6.4 min (red - peak highlighted), 2,4-DAB 333.01 m/z &#62; 273.10 m/z at 6.4 min (green), AEG 333.01 m/z &#62; 88.00 m/z at 7.4 min (orange), and BMAA 333.01 m/z &#62; 187.10 m/z at 7.8 min (blue). The dashed lines represent the retention times for missing 2,4-DAB, AEG, and BMAA. <a href="https://www.jove.com/files/ftp_upload/63779/63779fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: BMAA, AEG, and&#160;2,4-DAB concentrations in cyanobacterial isolates. Concentrations &#177; standard error of the mean (n = 3). ND denotes not detected. The highest concentration of each isomer detected is highlighted in green. The table is a modified version from Violi et al.38. <a href="https://www.jove.com/files/ftp_upload/63779/63779_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis at JoVE.com

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

The present protocol describes the extraction of non-protein amino acids from biological matrices via trichloroacetic acid (TCA) protein precipitation and acid hydrolysis before analysis using liquid chromatography-tandem mass spectrometry.

Non-protein amino acids (NPAAs) are a large class of amino acids (AAs) that are not genetically encoded for translation into proteins. The analysis of ...

extraction-non-protein-amino-acids-from-cyanobacteria-for-liquid

Research

JoVE Journal

Chemistry

3.6K Views.  University of Technology Sydney. Dit protocol zal gebruikers helpen en studenten de juiste stappen leren bij de extractie van BMA, AEG en 2, 4-DAB. Daardoor wordt de kans op fouten geminimaliseerd en consistente resultaten behaald. Deze methode is een veelgebruikte en gevalideerde methode voor de extractie van BMA en zijn isomeren, AEG en 2, 4-DAB in een sauna bacteriële matrix.Deze methode kan worden gebruikt om verder onderzoek te doen naar de toxiciteit van BMA's en een beter begrip te geven van BMA en de bijbehor...

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

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Biochemistry

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

Developmental Biology

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

Bioengineering

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Biology

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

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

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

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

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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

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