The authors thank Heather Roshon (Canadian Phycological Culture Centre, University of Waterloo for providing the cyanobacteria culture studied and Sawsan Abusharkh (Carleton University) for technical assistance.

1. Preparation of non-targeted liquid chromatography (LC)-MS/MS datasets
NOTE: DFF can be performed using any high-resolution mass spectrometer and analytical method optimized for a target class of analytes. MC optimized LC-MS/MS conditions on Orbitrap mass spectrometer are listed in the Table of Materials.
<ol>
	<li>Downloading MZmine 2 (http://mzmine.github.io/) 
	NOTE: Example data CPCC300.raw can be found at https://drive.google.com/open?id=1HHbLd...

<ol>
	<li>Mayer, P. M., Poon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+collisional+activation+of+ions+in+mass+spectrometry.">The mechanisms of collisional activation of ions in mass spectrometry.</a> Mass Spectrometry Reviews. 28 (4), 608-639 (2009).</li><li>Fisch, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+natural+products+by+microbial+iterative+hybrid+PKS&#8211;NRPS.">Biosynthesis of natural products by microbial iterative hybrid PKS&#8211;NRPS.</a> RSC Advances. 3 (40), 18228-18247 (2013).</li><li>Kelman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+six+new+Alternaria+sulfoconjugated+metabolites+by+high-resolution+neutral+loss+filtering.">Identification of six new Alternaria sulfoconjugated metabolites by high-resolution neutral loss filtering.</a> Rapid Communications in Mass Spectrometry. 29 (19), 1805-1810 (2015).</li><li>Renaud, J. B., Kelman, M. J., Qi, T. F., Seifert, K. A., Sumarah, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+ion+filtering+with+rapid+polarity+switching+for+the+detection+of+all+fumonisins+and+AAL-toxins.">Product ion filtering with rapid polarity switching for the detection of all fumonisins and AAL-toxins.</a> Rapid Communications in Mass Spectrometry. 29 (22), 2131-2139 (2015).</li><li>Renaud, J. B., Kelman, M. J., McMullin, D. R., Yeung, K. K. -. C., Sumarah, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+C8+liquid+chromatography-tandem+mass+spectrometry+for+the+analysis+of+enniatins+and+bassianolides.">Application of C8 liquid chromatography-tandem mass spectrometry for the analysis of enniatins and bassianolides.</a> Journal of Chromatography A. 1508, 65-72 (2017).</li><li>Walsh, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Fragmentation+Filtering+for+the+Discovery+of+New+Chaetoglobosins+and+Cytochalasins.">Diagnostic Fragmentation Filtering for the Discovery of New Chaetoglobosins and Cytochalasins.</a> Rapid Communications in Mass Spectrometry. , (2018).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharing+and+community+curation+of+mass+spectrometry+data+with+Global+Natural+Products+Social+Molecular+Networking.">Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.</a> Nature biotechnology. 34 (8), 828 (2016).</li><li>Bart&#243;k, T., Sz&#233;csi, &. #. 1. 9. 3. ;., Szekeres, A., Mesterh&#225;zy, &. #. 1. 9. 3. ;., Bart&#243;k, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+new+fumonisin+mycotoxins+and+fumonisin-like+compounds+by+reversed-phase+high-performance+liquid+chromatography/electrospray+ionization+ion+trap+mass+spectrometry.">Detection of new fumonisin mycotoxins and fumonisin-like compounds by reversed-phase high-performance liquid chromatography/electrospray ionization ion trap mass spectrometry.</a> Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up-to-the-Minute Research in Mass Spectrometry. 20 (16), 2447-2462 (2006).</li><li>Bart&#243;k, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+twenty-eight+isomers+of+fumonisin+B1+(FB1)+mycotoxin+in+a+solid+rice+culture+infected+with+Fusarium+verticillioides+by+reversed-phase+high-performance+liquid+chromatography/electrospray+ionization+time-of-flight+and+ion+trap+mass+spectrometry.">Detection and characterization of twenty-eight isomers of fumonisin B1 (FB1) mycotoxin in a solid rice culture infected with Fusarium verticillioides by reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight and ion trap mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 24 (1), 35-42 (2010).</li><li>Pluskal, T., Castillo, S., Villar-Briones, A., Ore&#353;i&#269;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MZmine+2:+modular+framework+for+processing,+visualizing,+and+analyzing+mass+spectrometry-based+molecular+profile+data.">MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data.</a> BMC bioinformatics. 11 (1), 395 (2010).</li><li>Spoof, L., Catherine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+3:+tables+of+microcystins+and+nodularins.">Appendix 3: tables of microcystins and nodularins.</a> Handbook of cyanobacterial monitoring and cyanotoxin analysis. , 526-537 (2016).</li><li>Pick, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blooming+algae:+a+Canadian+perspective+on+the+rise+of+toxic+cyanobacteria.">Blooming algae: a Canadian perspective on the rise of toxic cyanobacteria.</a> Canadian Journal of Fisheries and Aquatic Sciences. 73 (7), 1149-1158 (2016).</li><li>Carmichael, W. W., Boyer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+impacts+from+cyanobacteria+harmful+algae+blooms:+Implications+for+the+North+American+Great+Lakes.">Health impacts from cyanobacteria harmful algae blooms: Implications for the North American Great Lakes.</a> Harmful algae. 54, 194-212 (2016).</li><li>Mayumi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+microcystins+by+LC/MS/MS+under+ion+trap+conditions.">Structural characterization of microcystins by LC/MS/MS under ion trap conditions.</a> The Journal of antibiotics. 59 (11), 710 (2006).</li><li>Frias, H. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+electrospray+tandem+mass+spectrometry+for+identification+of+microcystins+during+a+cyanobacterial+bloom+event.">Use of electrospray tandem mass spectrometry for identification of microcystins during a cyanobacterial bloom event.</a> Biochemical and biophysical research communications. 344 (3), 741-746 (2006).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Watanabe, M. F., Oishi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+factors+on+toxicity+of+a+cyanobacterium+(Microcystis+aeruginosa)+under+culture+conditions.">Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions.</a> Applied and Environmental microbiology. 49 (5), 1342-1344 (1985).</li><li>Hollingdale, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+study+on+production+of+a+matrix+reference+material+for+cyanobacterial+toxins.">Feasibility study on production of a matrix reference material for cyanobacterial toxins.</a> Analytical and bioanalytical chemistry. 407 (18), 5353-5363 (2015).</li><li>Yuan, M., Namikoshi, M., Otsuki, A., Sivonen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+amino+acid+side-chain+on+fragmentation+of+cyclic+peptide+ions:+differences+of+electrospray+ionization/collision-induced+decomposition+mass+spectra+of+toxic+heptapeptide+microcystins+containing+ADMAdda+instead+of+Adda.">Effect of amino acid side-chain on fragmentation of cyclic peptide ions: differences of electrospray ionization/collision-induced decomposition mass spectra of toxic heptapeptide microcystins containing ADMAdda instead of Adda.</a> European Mass Spectrometry. 4 (4), 287-298 (1998).</li><li>Schymanski, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+small+molecules+via+high+resolution+mass+spectrometry:+communicating+confidence.">Identifying small molecules via high resolution mass spectrometry: communicating confidence.</a> Environmental science &#38; technology. 48 (4), 2097 (2014).</li></ol>

DFF is a straight-forward and rapid strategy for detecting entire classes of compounds, especially relevant for natural product compound discovery. The most important aspect of DFF is defining the specific MS/MS fragmentation criteria for the targeted class of compounds. In this representative example, DFF was used to detect all Adda residue containing MCs present in an M. aeruginosa cellular extract. Although the vast majority of MCs contain an Adda residue, other residues at this position have been known, nota...

Tandem mass spectrometry (MS/MS) is a widely used mass spectrometry method that involves isolating a precursor ion and inducing fragmentation via application of activation energy such as collision induced dissociation (CID)1. The manner in which an ion fragments is intimately linked to its molecular structure. Natural products are often biosynthesized as mixtures of structurally similar compounds rather than as a single unique chemical2. As such, structurally related compounds that are part of the same biosynthetic class often share key MS/MS fragmentation characteristics, including shared product ions and/or neutral losses. The ability to screen complex samples for compounds that possess class-specific product ions and/or neutral losses is a powerful strategy to detect entire classes of compounds, potentially leading to the discovery of new natural products3,4,5,6. For decades, mass spectrometry methods such as neutral loss scanning and precursor ion scanning performed on low resolution instruments have allowed ions with the same neutral loss or product ions to be detected. However, the specific ions or transitions needed to be defined prior to performing the experiments. As high-resolution mass spectrometers have become more popular in research laboratories, complex samples are now commonly screened using non-targeted, data-dependent acquisition (DDA) methods. In contrast to traditional neutral loss and precursor ion scanning, structurally related compounds can be identified by post-acquisition analysis7. In this work, we demonstrate a strategy we have developed termed diagnostic fragmentation filtering (DFF)5,6, a straight-forward and user-friendly approach to detect entire classes of compounds within complex matrices. This DFF module has been implemented into the open-source, MZmine 2 platform and available by downloading MZmine 2.38 or newer releases. DFF allows users to efficiently screen DDA datasets for MS/MS spectra which contain product ion(s) and/or neutral loss(es) that are diagnostic for entire classes of compounds. A limitation of DFF is characteristic product ions and/or neutral losses for a class of compounds must be defined by the analyst.
For example, each of the more than 60 different fumonisin mycotoxins identified8,9 possess a tricarballylic side chain, that generates a m/z 157.0142 (C6H5O5-) product ion upon fragmentation of the [M-H]- ion4. Therefore, all putative fumonisins in a sample can be detected using DFF by screening all MS/MS spectra within a DDA dataset that contain the prominent m/z 157.0142 product ion. Similarly, sulfated compounds can be detected by screening DDA datasets for MS/MS spectra that contain a diagnostic neutral loss of 79.9574 Da (SO3)3. This approach has also been successfully applied for detecting new cyclic peptides5 and natural products that contain tryptophan or phenylalanine residues6.
To demonstrate the effectiveness of DFF and its ease of use within the MZmine platform10, we have applied this approach to the analysis of microcystins (MCs); a class of over 240 structurally related toxins produced by freshwater cyanobacteria11,12,13.
The most commonly reported cyanotoxins are MCs, with the MC-LR (leucine [L]/arginine [R]) congener most frequently studied (Figure 1). MCs are monocyclic non-ribosomal heptapeptides, biosynthesized by multiple cyanobacteria genera including Microcystis, Anabaena, Nostoc, and Planktothrix12,13. MCs are composed of five common residues and two variable positions occupied by L-amino acids. Nearly all MCs possess a characteristic &#946;-amino acid 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) residue at position 511. &#160;The MS/MS fragmentation pathways of MCs are well described14,15; the Adda residue is responsible for the prominent MS/MS product ion, m/z 135.0803+ (C9H11O+) as well as other product ions including m/z 163.1114+ (C11H15O+) (Figure 2). Non-targeted DDA datasets of Microcystis aeruginosa cellular extracts can be screened for all microcystins present using these diagnostic ions, granted that the microcystins have an Adda residue.

<table>
	<tbody>
		<tr>
			<td>Cyanobacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcystis aeruginosaCPCC300</td>
			<td>CANADIAN PHYCOLOGICAL CULTURE CENTRE</td>
			<td>CPCC300</td>
			<td>https://uwaterloo.ca/canadian-phycological-culture-centre/</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteowizard (software)</td>
			<td></td>
			<td>software</td>
			<td>http://proteowizard.sourceforge.net/</td>
		</tr>
		<tr>
			<td>Mzmine 2</td>
			<td></td>
			<td>software</td>
			<td>http://mzmine.github.io/</td>
		</tr>
		<tr>
			<td>LC-MS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Q-Exactive Orbitrap</td>
			<td>Thermo</td>
			<td>-</td>
			<td>Equipped with HESI ionization source</td>
		</tr>
		<tr>
			<td>1290 UHPLC</td>
			<td>Agilent</td>
			<td></td>
			<td>Equipped with binary pump, autosampler, column compartment</td>
		</tr>
		<tr>
			<td>C18 column</td>
			<td>Agilent</td>
			<td>959757-902</td>
			<td>Eclipse Plus C18 RRHD column (2.1 &#215; 100 mm, 1.8 &#956;m)</td>
		</tr>
		<tr>
			<td>Solvents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima LC-MS grade Methanol</td>
			<td>Fisher</td>
			<td>A456-4</td>
			<td></td>
		</tr>
		<tr>
			<td>OptimaLC-MS grade Acetonitrile</td>
			<td>Fisher</td>
			<td>A955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>OptimaLC-MS grade Water</td>
			<td>Fisher</td>
			<td>W6-4</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS grade Formic Acid</td>
			<td>Fisher</td>
			<td>A11710X1-AMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Sorvall Micro 21</td>
			<td>Thermo Scientific</td>
			<td>75-772-436</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amber HPLC vials 2 mL/caps</td>
			<td>Agilent</td>
			<td>5182-0716/5182-0717</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2-&#956;m PTFE syringe filters</td>
			<td>Pall Corp.</td>
			<td>4521</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman 47mm GF/A glass microfiber filters</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1820047</td>
			<td></td>
		</tr>
		<tr>
			<td>Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MA media (pH 8.6) ( quantity / L)</td>
			<td></td>
			<td></td>
			<td>Watanabe, M. F. &#38; Oishi, S. Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions. Applied and Environmental microbiology. 49 (5), 1342-1344 (1985).</td>
		</tr>
		<tr>
			<td>Ca(NO3)&#183;4H2O, 50 mg</td>
			<td>Sigma-Aldrich</td>
			<td>C2786</td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3, 100 mg</td>
			<td>Sigma-Aldrich</td>
			<td>P8291</td>
			<td></td>
		</tr>
		<tr>
			<td>NaNO3, 50 mg</td>
			<td>Sigma-Aldrich</td>
			<td>S5022</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2SO4, 40 mg</td>
			<td>Sigma-Aldrich</td>
			<td>S5640</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H20, 50 mg</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium glycerophosphate, 100 mg</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>H3BO3, 20 mg</td>
			<td>Sigma-Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicine, 500 mg</td>
			<td>Sigma-Aldrich</td>
			<td>RES1151B-B7</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>P(IV) metal solution, 5 mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bring the following to 1 L with ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaEDTA&#183;2HO</td>
			<td>Sigma-Aldrich</td>
			<td>E6635</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3 &#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2&#183;4H2O</td>
			<td>Baker</td>
			<td>2540</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>Z0152</td>
			<td></td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2MoO4&#183;2H2O</td>
			<td>Baker</td>
			<td>3764</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanobacteria BG-11 50X Freshwater Solution</td>
			<td>Sigma-Aldrich</td>
			<td>C3061-500mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The DFF plot generated following the analysis of M. aeruginosa CPCC300 is shown in Figure 4. The x-axis of this plot is the m/z of the precursor ions that satisfied the defined DFF criteria while the y-axis shows the m/z of all product ions within the MCs MS/MS spectra. For this analysis, the criteria for MC detection included precursor ions within the m/z range of 440-1200, retention times between 2.00&#8...

Watch this Scientific Journal Video about Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis at JoVE.com

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

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 ...

Natural products are often biosynthesized as a class of compounds, and not as a single product. Diagnostic fragmentation filtering is a simple approach for detecting all structurally-related compounds within a complex mixture. Diagnostic fragmentation filtering, implemented within the MZmine software, allows the analyst to explore mass spectrometry data sets for new natural products that would otherwise be missed.This includes new bioproducts, and also new toxins. Diagnostic fragmentation filtering, applied to toxin classes that contaminate food and water, can inform exposure assessments. When applied to microbial and plant extracts, it can result in the discovery of new compounds.Using diagnostic fragmentation filtering requires an understanding of the key tandem mass spectrometry features that define a chemical class. This means that structurally similar compounds will generate characteristic ions. My name is Shawn Hoogstra.I am a research technician and software developer at our center, and I will be demonstrating the procedure. To prepare a sample for microcystin analysis, inoculate 30 milliliters of sterile cyanobacteria growth medium to approximately five-times-10-to-the-fifth cells per milliliter, under aseptic conditions, in 250-milliliter Erlenmeyer flasks, monitoring the cell density with a hemocytometer. After 26 days of photoautotrophic culture, illuminated with cool white fluorescent light using a 12-hour light-dark regime at 27 degrees Celsius, with swirling once daily, use 47-milliliter diameter GF/C glass microfiber filter papers to separate the cells from the culture medium by vacuum filtration.Add three milliliters of 80%methanol to the harvested cells in 14-milliliter test tubes, and vortex and subsequently sonicate each tube for 30 seconds. After sonication, lyse the samples with three consecutive one-hour, minus-20-degree-Celsius freeze, and 15-minute room temperature thaw cycles, and filter each resulting cyanobacteria cell extract through individual 0.22-micrometer polytetrafluoroethylene syringe filters. Using a gentle stream of nitrogen gas, dry the extracts with an evaporator at 30 degrees Celsius, and store the extracts dry at minus-20 degrees Celsius.For liquid chromatography tandem mass spectrometry analysis, reconstitute the dried residue to be analyzed with 500 microliters of 90%methanol, and vortex the samples for 30 seconds. Transfer to an amber high-pressure liquid chromatography vial. Then analyze the cyanobacteria extract using a data-dependent acquisition method on a high-resolution mass spectrometer according to standard protocols.Most microcystins contain an ATA residue that produces two diagnostic product ions in tandem mass spectrometry, M through Z 135.0803, and 163.1114. For non-targeted liquid chromatography tandem mass spectrometry data set preparation, open MZmine 2, and select Raw data import"under Raw data methods"drop-down menu. Then select the data files from the microcystin analysis, and select the peak picking filter to apply a vendor-supplied centroiding algorithm.For diagnostic fragmentation filtering, or DFF, of the imported data-dependent acquisition files, use the cursor to highlight the data files, and open the Visualization"drop-down menu to select the DFF option. In the DFF dialogue box that appears, input the range of retention times and minutes when the targeted class of analytes will elute, and to find the mass-to-charge ratio range of the targeted class of analytes, including the possibility for multiple charged compounds when appropriate. Input the achievable tandem mass spectrometry mass accuracy of the mass spectrometry instrument, and input the class-specific product ions of the mass-to-charge ratio.Enter the class-specific neutral losses, and define the minimum intensity for diagnostic product ions and/or neutral losses to be considered as a percentage of the base peak of the tandem mass spectrometry spectra. Then select a path and file name to output the results and click OK"to start the DFF. A DFF plot will appear upon a successful completion of the setup.The analyst must interpret tandem mass spectra of known compounds within a compound class to identify the product ions and/or neutral losses that are diagnostic for the entire class of compounds. This DFF plot was generated following the analysis of M.aeruginosa CPCC 300. The x-axis represents this mass-to-charge ratio of the precursor ions that satisfied the defined DFF criteria, while the y-axis shows the mass-to-charge ratio of all of the product ions within the microcystins tandem mass spectrometry spectra.For this analysis, the criteria for microcystin detection included precursor ions within the mass-to-charge ratio of 400 to 1, 200, and retention times between two to six minutes. Most importantly, these tandem mass spectrometry spectra contained both mass-to-charge ratios above the defined 15%base peak intensity threshold. Of the 4, 116 tandem mass spectrometry spectra acquired during the analysis, 26 satisfied the DFF criteria and were detected in the M.aeruginosa CPCC 300 extract.The major microcystins detected could be confidently assigned as microcystin leucine arginine and demethylated aspartic acid microcystin leucine arginine. As new compounds are discovered using diagnostic fragmentation filtering, the organisms can be grown in large scale for isolation and characterization of the compounds by NMR. When applied to microcystins, diagnostic fragmentation filtering allows us to determine if traditional targeted screening methods are detecting the major toxins present within a sample.

natural-product-discovery-with-lc-msms-diagnostic-fragmentation

Research

JoVE Journal

Chemistry

8.6K Views.  Carleton University Ottawa. I prodotti naturali sono spesso biosintesi come una classe di composti, e non come un singolo prodotto. Il filtraggio diagnostico della frammentazione è un approccio semplice per rilevare tutti i composti strutturalmente correlati all'interno di una miscela complessa. Il filtro a frammentazione diagnostica, implementato all'interno del software MZmine, consente all'analista di esplorare set di dati di spettrometria di massa per nuovi prodotti naturali che altrimenti sarebbero mancati....