Name: natural product discovery with lc-ms/ms diagnostic fragmentation filtering: application for microcystin analysis
Uploaded: 2019-05-31T14:00:00
Description: Watch this Scientific Journal Video about Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis at JoVE.com

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

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

A Two-Step Pyrolysis-Gas Chromatography Method with Mass Spectrometric Detection for Identification of Tattoo Ink Ingredients and Counterfeit Products

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. In this method for two-step pyrolysis online coupled to gas chromatography mass spectrometry (py-GC-MS) volatile compounds are analyzed during a first desorption run. In the second run, the same dried sample is pyrolyzed for analysis of non-volatile compounds such as pigments and polymers. These can be identified by their specific decomposition patterns. Additionally, this method can be used to differentiate original from counterfeit inks. Easy screening methods for data evaluation using the average mass spectra and self-made pyrolysis libraries are applied to speed up substance identification. Using specialized evaluation software for pyrolysis GS-MS data, a fast and reliable comparison of the full chromatogram can be achieved. Since GC-MS is used as separation technique, the method is limited to volatile substances upon desorption and after pyrolysis of the sample. The method can be applied for quick substance screening in market control surveys since it requires no sample preparation steps.

This method for two-step pyrolysis online coupled to gas chromatography with mass spectrometric detection and data evaluation protocol can be used for multi-component analysis of tattoo inks and discrimination of counterfeit products.

Tattoo inks are complex mixtures of ingredients. Each of them possesses different chemical properties which have to be addressed upon chemical analysis. ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

A number of different procedures for preparing nanoparticles for surface analysis are presented (drop casting, spin coating, deposition from powders, and cryofixation). We discuss the challenges, opportunities, and possible applications of each method, particularly regarding the changes in the surface properties caused by the different preparation methods.

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...

ARL Spectral Fitting as an Application to Augment Spectral Data via Franck-Condon Lineshape Analysis and Color Analysis

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis (FCLSA) on spectral data, in addition to CIE color coordinate determination and basic spectral processing. While some of the features may be found in commercial software or in programs made by&#160;academic research groups, we believe that ARL Spectral Fitting is the only application that possesses all three of the aforementioned attributes.
This program is intended as a standalone, GUI-based application for use by an average laboratory researcher without requiring any coding knowledge or proprietary software. In addition to the standalone executable hosted on ARL GitHub, the associated MATLAB files are available for use and further development.
FCLSA augments the information found in luminescence spectra, providing meaningful insight into the relationship between the ground and excited states of a photoluminescent species. This insight is achieved by modeling spectra with two versions (modes) of an equation that are characterized by either four or six parameters, depending on which mode is used. Once optimized, the value of each of these parameters can be used to gain insight into the molecule, as well as to perform further analysis (for example, the free energy content of the excited-state molecule). This application provides tools for easy by-hand fitting of imported data, as well as two methods for optimizing this fit-damped least-squares fitting, powered by the Levenberg-Marquardt algorithm, and derivative-free fitting utilizing the Nelder-Mead simplex algorithm. Furthermore, estimations of sample color can be performed and reported in CIE and RGB coordinates.

This protocol introduces Franck-Condon Lineshape Analyses (FCLSA) of emission spectra and serves as a tutorial for the use of ARL Spectral Fitting software. The open-source software provides an easy and intuitive way to perform advanced analysis of emission spectra including excited state energy calculations, CIE color coordinate determination, and FCLSA.

The ARL Spectral Fitting application provides a free, publicly accessible, and fully transparent method for performing Franck-Condon Lineshape Analysis ...