The financial support for this study was provided by S&#227;o Paulo Research Foundation - FAPESP (2019/10564-5, 2014/12727-5 and 2014/50249-8 to L.G.O; 2013/12598-8 and 2015/01013-4 to R.S.; and 2019/08853-9 to C.F.F.A). B.S.P, C.F.F.A., and L.G.O. received fellowships from the National Council for Scientific and Technological Development - CNPq (205729/2018-5, 162191/2015-4, and 313492/2017-4). L.G.O. is also grateful for the grant support provided by the program For Women in Science (2008, Brazilian Edition). All authors acknowledge CAPES (Coordination for the Improvement of Higher Education Personnel) for supporting the post-graduation programs in Brazil.

1. Genome mining for biosynthetic gene clusters
<ol>
	<li>Perform whole genome sequencing (WGS) as the first step to electing a biosynthetic gene cluster (BCG) for MS-guided genome mining. The whole genome draft of the strain of interest (bacteria) can be obtained by Illumina MiSeq technology using the following with high quality genomic DNA: shotgun TruSeq PCR-Free library prep and Nextera Mate Pair Library Preparation Kit33. 
	NOTE: After sequencing, the Illumina shotgun library and Illumina mate pair library can be assembled using the Newbler v3.0 (Roche, 454) assembler program (found at &#60;https://ngs.csr.uky.edu/Newbler&#62;) and annotated using a pipeline based on FgeneSB (found at &#60;http://www.softberry.com/berry.phtml?topic=fgenesb_annotator&#38;group=help&#38;subgroup=pipelines&#62;), as described previously33. Microbiology Resource Announcements (MRA) is a fully open access journal with articles publishing the availability of any microbiological resource deposited in an available repository (found at &#60;https://mra.asm.org&#62;). The candidate protein-coding genes are identified using the RAST server annotation34, and the Whole Genome Shotgun (WGS) project is deposited in the DDBJ/ENA/GenBank (found at &#60;https://www.ncbi.nlm.nih.gov/genbank/&#62;) and Gold (found at &#60;https://gold.jgi.doe.gov&#62;) sequence databases.</li>
	<li>To obtain in silico information about secondary metabolism gene clusters annotations from a complete sequenced genome, submit the sequence file (GenBank/EMBL or FASTA format) to an antiSMASH platform (found at &#60;https://antismash.secondarymetabolites.org/&#62;).</li>
	<li>Select the gene cluster of interest from output data (Figure 2) based on the most similar known cluster. 
	NOTE: First, it is routine to explore gene-by-gene and conduct individual searches (blastp) to evaluate which functions are associated with the desired biosynthetic gene groups. This procedure can also help to determine which BGC is likely associated with the production of a desired compound, even if it is a low percentage. An antiSMASH prediction considers all genes within a cluster to make percentage coverage, which can represent a global low percentage of similarity for the aimed BGC. However, when analyzing gene-by-gene, it is possible to obtain more accurate information using the most similar known cluster. Second, antiSMASH has two options to refine a search: 1) detection strictness: the degree of strictness to which the biosynthetic gene cluster must be to be considered a hit. For this option, the user should use the following parameters: a) strict: detects exclusively well-defined clusters containing all required regions, insusceptible to errors about genetic information; b) relaxed: detects partial clusters missing one or more functional region, which also works for detecting the strict feature; or c) loose: detects poorly defined clusters and clusters that likely match primary metabolites, which can lead to appearance of false positives or poorly defined BGCs. The other option is 2) extra features: the type of information the platform must search for and show in the output. In general, these two options can save time after the prediction. However, the antiSMASH job requires a longer time period.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60825/60825fig2.jpg" /> 
Figure 2: Output from antiSMASH platform. Secondary metabolism in silico analysis from whole genome sequence annotation. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Based on DNA sequence information of the BGC, design primers (20&#8211;25 nt) flanking the gene cluster for ESAC (E. coli/Streptomyces Artificial Chromosome) library screening. 
	NOTE: Different methods35,36 can be used to capture the whole biosynthetic gene cluster from DNA. Here, the method used is construction of a representative ESAC library37,38 from Streptomyces sp. CBMAI 2042 containing clones with average size fragments of ~95 kb.</li>
</ol>
2. Heterologous expression of whole biosynthetic gene cluster from the ESAC library
<ol>
	<li>Move the ESAC vector from E. coli DH10B to E. coli ET12567 by triparental conjugation32.
	<ol>
		<li>Inoculate E. coli ET12567 (CamR), TOPO10/pR9604 (CarbR), DH10B/ESAC4H (AprR) in 5 mL of Luria-Bertani (LB) medium containing chloramphenicol (25 &#181;g/mL), carbenicillin (100 &#181;g/mL), and apramycin (50 &#181;g/mL).</li>
		<li>Incubate the culture overnight at 37 &#176;C and 250 rpm.</li>
		<li>Inoculate 500 &#181;L of the overnight culture in 10 mL of LB medium containing a half-concentration of antibiotics.</li>
		<li>Incubate the culture at 37 &#176;C and 250 rpm until reaching an A600 of 0.4&#8211;0.6.</li>
		<li>Harvest the cells by centrifugation at 2,200 x g for 5 min.</li>
		<li>Wash the cells twice with 20 mL of LB medium.</li>
		<li>Resuspend the cells in 500 &#181;L of LB medium.</li>
		<li>Mix 20 &#956;L of each strain in a microcentrifuge tube and drip into an agar plate with LB medium lacking antibiotics.</li>
		<li>Incubate the plates at 37 &#176;C overnight.</li>
		<li>Streak the grown cells onto a fresh LB agar plate containing antibiotics and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
</ol>
3. Streptomyces/E. coli conjugation
<ol>
	<li>To obtain the recombinant heterologous organism, perform conjugation32 between E. coli ET12567 containing the ESAC vector, helper plasmid pR9604, and Streptomyces coelicolor M1146 or another selected host strain39.</li>
	<li>Day 1: Inoculate isolated colonies of S. coelicolor M1146 in 25 mL of TSBY medium in a 250 mL Erlenmeyer flask fitted with an inox-spring at 30 &#173;&#176;C and 200 rpm for 48 h.</li>
	<li>Day 2/3: Inoculate ET12567/ESAC/pR9604 in 5 mL of LB medium containing chloramphenicol (25 &#181;g/mL), carbenicillin (100 &#181;g/mL), and apramycin (50 &#181;g/mL) overnight at 37 &#176;C and 250 rpm.</li>
	<li>Day 3/4: Inoculate 500 &#181;L of the overnight culture in 10 mL of 2TY (in a 50 mL conical tube) containing half-working concentrations of antibiotics. Incubate at 37 &#176;C and 250 rpm until reaching an A600 of 0.4&#8211;0.6.</li>
	<li>Centrifuge the cultures (ET12567/ESAC/pR9604 and M1146) at 2200 x g for 10 min.</li>
	<li>Wash the pellets 2x in 20 mL of 2TY medium and resuspend in 500 &#181;L of 2TY.</li>
	<li>Aliquot 200 &#181;L of the S. coelicolor M1146 suspension and dilute in 500 &#181;L of 2TY (suspension A).</li>
	<li>Aliquot 200 &#181;L of suspension A and dilute in 500 &#181;L of 2TY (suspension B).</li>
	<li>Aliquot 200 &#181;L of suspension B and dilute in 500 &#181;L of 2TY (suspension C).</li>
	<li>Aliquot 200 &#181;L of the ET12567/ESAC/pR9604 suspension and mix with 200 &#181;L of suspension C.</li>
	<li>Plate 150 &#181;L of the conjugation mixture on an SFM agar plate lacking antibiotics.</li>
	<li>Incubate at 30 &#176;C for 16 h.</li>
	<li>Cover plates with 1 mL of antibiotic solution (according to plasmid resistance). After drying, incubate at 30 &#176;C for 4&#8211;7 days. 
	NOTE: Here, a solution containing 1.0 mg/mL thiostrepton and 0.5 mg/mL nalidixic acid was prepared.</li>
	<li>Streak putative exconjugants onto SFM agar plates containing thiostrepton (50 mg/mL) and nalidixic acid (25 mg/mL). Incubate at 30 &#176;C.</li>
	<li>Streak exconjugants onto an SFM agar containing only nalidixic acid.</li>
	<li>Perform PCR analysis with isolated colonies to confirm that the entire gene cluster has been transferred to the S. coelicolor M1146 host.</li>
</ol>
4. Strain cultivation
<ol>
	<li>To obtain the metabolic profile, inoculate 1/100 of the strain&#39;s pre-culture in appropriate fermentation media and under the appropriate culture conditions.</li>
	<li>Centrifuge cultures at 2200 x g for 10 min.</li>
	<li>Perform the extraction according to the class of the compound of interest40.</li>
</ol>
5. Acquiring mass spectra and preparation for GNPS analysis
<ol>
	<li>To acquire MS/MS data, program suitable HPLC and mass spectrometry methods using the control software. Both high and low resolution data-dependent mass spectrometry analysis (DDA) can be analyzed. 
	NOTE: Generally, a 1 mg/mL solution of complex crude extract samples is ideal. Dilutions are needed for less complex extracts. It should be noted that MS/MS networking is the detectable molecular network under the given mass spectrometric conditions.</li>
	<li>Convert mass spectra to .mzXML format using MSConvert from Proteowizard (found at &#60;http://proteowizard.sourceforge.net/&#62;). The input parameters for the conversion are illustrated in Figure 3. Data from software of almost all companies are compatible.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60825/60825fig3.jpg" /> 
Figure 3: Using MsConvert to convert MS files to mzXML extension. The correct parameter for GNPS analysis is displayed. The instructions are as follows: add all MS files in box 1 and add the filter Peak Picking in box 2; for this filter, use the algorithm vendor; press start and the processes of conversion will follow. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Upload the converted LC-MS/MS files into the GNPS database. Two options are available: using a file transfer protocol (FTP) or directly in a browser through the online platform. 
	NOTE: Detailed information on how to install and transfer data to GNPS is available at &#60;https://ccms-ucsd.github.io/GNPSDocumentation/fileupload/&#62;.</li>
</ol>
6. GNPS analysis
<ol>
	<li>After creating an account in GNPS (found at &#60;https://gnps.ucsd.edu/&#62;), log in to the created account select Create Molecular Network. Add a job title.</li>
	<li>Basic options: select the mzXML files to perform the molecular network. They can be organized into up to six groups. Select the libraries for the dereplication routine (Figure 4). 
	NOTE: These groups do not interfere with molecular network construction. This information will be used only for the graphical representation.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60825/60825fig4b.jpg" /> 
Figure 4: Using online GNPS platform to perform molecular network analysis. Selection of mzXML files is done by clicking in box 1. In the open dialog box, the files can be selected from personal folder (box 2) or be uploaded in the second tab using the drag-and-drop file uploader (less than 20 MB). The files can be grouped into up to six groups. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Select the precursor ion mass tolerance and fragment ion mass tolerance of 0.02 Da and 0.05 Da, respectively. 
	NOTE: GNPS has different types of strictness available based on 1) how accurate the MS/MS data is and 2) how accurate the association must be. Basic options: in this folder, it is possible to set Precursor Ion Mass Tolerance and Fragment Ion Mass Tolerance. These parameters are used as a guide to determine how precise the precursor ion and fragment ion must be. The selected mass tolerances depend on the resolution and accuracy of the mass spectrometer that is used.</li>
	<li>Advanced network options: select the parameters according to Figure 5. These parameters directly influence the network cluster size and form. Another parameter in the remaining tabs section are for advanced users; thus, leave the default values. 
	NOTE: Advanced parameters can be read in GNPS documentation (found at &#60;https://ccms-ucsd.github.io/GNPSDocumentation/&#62;).</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60825/60825fig5.jpg" /> 
Figure 5: Using GNPS to perform molecular network analysis (advanced options). Min Pair Cos will directly influence the size of clusters, as high values will result in combining closely-related compounds and low values in combining distantly-related compounds. Using values that are too low should be avoided. Minimum matched fragment ions represent the number of shared fragments between two fragmentation spectra to be linked in the network. Together, both parameters guide the network format; lower values will cluster more distantly-related compounds and vice-versa. Using the proper values will greatly help the compound elucidation. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Choose an e-mail address to receive an alert when the work is done, and submit the job.</li>
</ol>
7. Analysis of GNPS results
<ol>
	<li>Log in to GNPS. Select Jobs &#62; Published job &#62; Done to open the job. A webpage will open as illustrated in Figure 6. All results obtained from molecular networking will be displayed.</li>
	<li>Select View Spectral Families (In Browser Network Visualizer) to see all network clusters (red box, Figure 6).</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60825/60825fig6.jpg" /> 
Figure 6: Using GNPS to visualize molecular network results. All related compound clusters can be seen in view spectral families (red box). To visualize only library hits, &#34;view all library hits&#34; (blue box) should be selected. For better graphical representation of molecular network results, &#34;Direct Cytoscape Preview&#34; (yellow box) should be downloaded, and the latest version of Cytoscape should be used. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>A list will be displayed with all generated molecular networking clusters. If a library search was selected to generate the findings, tentative molecules identification will be displayed in AllIDs. Select Show to visualize them. 
	NOTE: The data analyses can be driven for other results (i.e., genome mining, biological assays, library dereplication molecules, etc.).</li>
	<li>To analyze the molecular network cluster, select Visualize Network. 
	NOTE: Each cluster is composed of nodes (circles) and edges, which represents molecules and molecular similarity, respectively. Dereplicated molecules will be highlighted as a blue node in the online browser network visualizer.</li>
	<li>In the node labels box, select parent mass (red box, Figure 7).</li>
	<li>In the edge labels box, select Cosine or DeltaMZ to observe node similarity or mass difference between nodes, respectively (yellow box, Figure 7).</li>
	<li>In the case of multigroup analyses, click Draw pies in the node coloring box to observe the frequency at which each node appears in each group (blue box, Figure 7). 
	NOTE: Other choices are possible, but those suggested above are optimal for annotating cluster nodes and unraveling their structures.</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60825/60825fig7a.jpg" /> 
Figure 7: Using GNPS to visualize molecular cluster results. After opening the molecular clusters for better data visualization, the following should be chosen: &#34;Parent mass&#34; as node labels (red box); &#34;DeltaMZ&#34; as edge labels (yellow box); and &#34;Draw pies&#34; as node coloring (blue box). Navigate through the molecular cluster and try to annotate all nodes. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="8">
	<li>To see all library hits, select View all library hits (blue box, Figure 7). 
	NOTE: Also, the MNW can be downloaded in &#34;Direct Cytoscape Preview/Download&#34; (yellow box, Figure 7), and the file can be opened in the Cytoscape platform (found at &#60;https://cytoscape.org/&#62;) for more options in graphical structure.</li>
	<li>Manual confirmation of dereplicated compounds and structure elucidation of related compounds are needed. Open the fragmentations spectra directly in the GNPS platform or in original raw files.</li>
</ol>

The authors have nothing to disclose.

The strongest advantage of this protocol is its ability to rapidly dereplicate metabolic profiles and bridge genomic information with MS data in order to elucidate the structures of new molecules, especially structural analogues2. Based on genomic information, different natural products chemotypes can be investigated, such as polyketides (PK), nonribosomal peptides (NRP), and glycosylated natural products (GNP), as well as cryptic BGCs. Metabolomic screening yields evidence of activated BGC profil...

Investigations of secondary metabolism often consist of screening crude extracts for specific biological activities followed by purification, identification, and characterization of the constituents belonging to active fractions. This process has proved to be efficient, promoting the isolation of several chemical entities. However, nowadays this is seen as unfeasible, mainly due to the high rates of rediscovery. As the pharmaceutical industry revolutionized without knowledge of the roles and functions of specialized metabolites, their identification was carried out under laboratory conditions that did not accurately represent nature1. Today, there is a better understanding of natural signaling influences, secretion, and the presence of most targets at undetectably low concentrations. Additionally, regulation of the process will help the academic community and pharmaceutical industry to take advantage of this knowledge. It will also benefit research involving the direct isolation of metabolites related to silent biosynthetic gene clusters (BGCs)2.
In this context, advances in genomic sequencing have renewed interest in screening microorganism metabolites. This is because analyzing the genomic information of uncovered biosynthetic clusters can reveal genes encoding novel compounds not observed or produced under laboratory conditions. Many microbial whole genome projects or drafts are available today, and the number is growing every year, providing massive prospects for uncovering novel bioactive molecules through genome mining3,4.
The Atlas of Biosynthetic Gene Clusters is the current largest collection of automatically mined gene clusters as a component of the Integrated Microbial Genomes Platform of the Joint Genome Institute (JGI IMG-ABC)2. Most recently, the Minimum Information for Biosynthetic Gene Clusters (MIBiG) Standardization Initiative has promoted the manual reannotation of BGCs, providing a highly curated reference dataset5. Nowadays, plenty of tools are available to enable computational mining of genetic data and their connection to known secondary metabolites. Different strategies have also been developed to access new bioactive natural products (i.e., heterologous expression, target gene deletion, in vitro reconstitution, genomic sequence, isotope-guided screening [genomisotopic approach], manipulation of local and global regulators, resistance target-based mining, culture independent mining, and, more recently, MS-guided/code approaches2,6,7,8,9,10,11,12,13,14,15).
Genome mining as a singular strategy requires efforts to annotate a single or small group of molecules; thus, gaps in the process remain in which new compounds are prioritized for isolation and structure elucidation. In principle, these approaches target only one biosynthetic pathway per experiment, thereby resulting in a slow discovery rate. In this sense, using GM along with a molecular networking approach represents an important advance for natural product research14,15.
The versatility, accuracy, and high sensitivity of liquid chromatography-mass spectrometry (LC-MS) make it a good method for compound identification. Currently, several platforms have invested algorithms and software suites for untargeted metabolomics16,17,18,19,20. The core of these programs includes feature detection (peak picking)21 and peak alignment, which allows match of identical features across a batch of samples and searching for patterns. MS pattern-based algorithms22,23 compare characteristic fragmentation patterns and match MS2 similarities generating molecular families sharing structural features. These features can then be highlighted and clustered, conferring the ability to rapidly discover known and unknown molecules from a complex biological extract by tandem MS2,24,25. Therefore, tandem MS is a versatile method to gain structural information of several chemotypes contained in a large amount of data simultaneously.
The Global Natural Products Social Molecular Networking (GNPS)26 algorithm uses the normalized fragment ions intensity to construct multidimensional vectors, in which similarities are compared using a cosine function. The relationship between different parent ions are plotted in a diagram representation, in which each fragmentation is visualized as a node (circles), and the relatedness of each node is defined by an edge (lines). The global visualization of molecules from a single source is defined as a molecular network. Structurally divergent molecules that fragment uniquely will form their own specific cluster or constellation, whereas related molecules cluster together. Clustering chemotypes allows the hypothetical connection of similar structural features to their biosynthetic origins.
Combining both chemotype-to-genotype and genotype-to-chemotype approaches is powerful when creating bioinformatics links between BGCs and their small molecule products27. Therefore, MS-guided genome mining is a rapid method and low material-consuming strategy, and it helps bridge parent ions and biosynthetic pathways revealed by WGS of one or more strains under diverse metabolic and environmental conditions.
The workflow of this protocol (Figure 1) consists of feeding WGS data into a biosynthetic gene cluster annotation platform such as antiSMASH28,29,30. It helps estimate the variety of compounds and class of compounds encoded by the genome. A strategy to target a biosynthetic gene cluster encoding a chemical entity of interest must be adopted, and culture extracts from a wild type strain and/or heterologous strain containing the BGC can be analyzed to generate clustered ions based on similarities using GNPS26,31. Consequently, it is possible to identify new molecules that associate with the targeted BGC and are unavailable in the database (mainly unknown analogues, sometimes produced in low titers). It is relevant to consider that users can contribute to these platforms and that the availability of bioinformatics and MS/MS data is increasing rapidly, driving to a constant development and upgrade of effective computational tools and algorithms to guide efficient connections of complex extracts with molecules.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60825/60825fig1b.jpg" /> 
Figure 1: Overview of the entire workflow. Shown is an illustration of the bioinformatic, cloning, and molecular networking steps involved in the described MS-guided genome mining approach to identify new metabolites. <a href="https://www.jove.com/files/ftp_upload/60825/60825fig1blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This protocol describes a rapid and efficient workflow to combine genome mining and molecular networking as starting point for the natural product discovery pipeline. Although many applications are able to visualize the composition and relatedness of MS-detectable molecules in one network, several are adopted here to visualize structurally similar clustered molecules. Using this strategy, novel cyclodepsipeptide products observed in metabolic extracts of Streptomyces sp. CBMAI 2042 are successfully identified. Guided by genome mining, the whole biosynthetic gene cluster encoding for valinomycins is recognized and cloned into the producer strain Streptomyces coelicolor M1146. Finally, following a MS pattern-based molecular networking, the molecules detected by MS are correlated with BGCs responsible for their biogenesis32.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Tedia</td>
			<td>AA1120-048</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Oxoid</td>
			<td>LP0011</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Apramycin</td>
			<td>Sigma Aldrich</td>
			<td>A2024</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Sigma Aldrich</td>
			<td>C9231</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>NA</td>
			<td>5804</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma Aldrich</td>
			<td>C3175</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Column C18</td>
			<td>Agilent Technologies</td>
			<td>NA</td>
			<td>ZORBAX RRHD Extend-C18, 80&#197;, 2.1 x 50 mm, 1.8 &#181;m, 1200 bar pressure limit P/N 757700-902</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma Aldrich</td>
			<td>K1377</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Manitol P.A.- A.C.S.</td>
			<td>Synth</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>NA</td>
			<td>5418</td>
		</tr>
		<tr>
			<td>Nalidixic acid</td>
			<td>Sigma Aldrich</td>
			<td>N4382</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Phusion Flash High-Fidelity PCR Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td>F548S</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Q-TOF mass spectrometer</td>
			<td>Agilent technologies</td>
			<td>NA</td>
			<td>6550 iFunnel Q-TOF LC/MS</td>
		</tr>
		<tr>
			<td>Sacarose P.A.- A.C.S.</td>
			<td>Synth</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Shaker/Incubator</td>
			<td>Marconi</td>
			<td>MA420</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Synth</td>
			<td>NA</td>
			<td>P. A. - ACS</td>
		</tr>
		<tr>
			<td>Soy extract</td>
			<td>NA</td>
			<td>NA</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Synth</td>
			<td>NA</td>
			<td>P. A. - ACS</td>
		</tr>
		<tr>
			<td>Thermal Cycles</td>
			<td>Eppendorf</td>
			<td>NA</td>
			<td>Mastercycler Nexus Gradient</td>
		</tr>
		<tr>
			<td>Thiostrepton</td>
			<td>Sigma Aldrich</td>
			<td>T8902</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Tryptone Soy Broth</td>
			<td>Oxoid</td>
			<td>CM0129</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>UPLC</td>
			<td>Agilent Technologies</td>
			<td>NA</td>
			<td>1290 Infinity LC System</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>

mass spectrometry-guided genome mining as a tool to uncover novel natural products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

The protocol was successfully exemplified using a combination of genome mining, heterologous expression, and MS-guided/code approaches to access new specialized valinomycin analogues molecules. The genome-to-molecule workflow for the target, valinomycin (VLM), is represented in Figure 8. Streptomyces sp. CBMAI 2042 draft genome was analyzed in silico, and the VLM gene cluster was then identified and transferred to a heterologous host. Heterologous and wild type strains were cultivat...

Watch this Scientific Journal Video about Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products at JoVE.com

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...

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Chemistry

10.8K Views.  University of Campinas (UNICAMP). A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

Video: Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment allows for testing of various proposed ocean / hydrothermal fluid chemistries that could have driven reactions toward the origin of life in environments on the early Earth, early Mars, or even other worlds such as the icy moons of the outer planets. We show experiments that include growth of chemical garden structures under anoxic conditions simulating the early Earth, inclusion of trace components of phosphates / organics in the injection solution to incorporate them into the structure, a switch of the injection solution to introduce a secondary precipitating anion, and the measurement of membrane potentials generated by chemical gardens. Using this method, self-assembling chemical garden structures were formed that mimic the natural chimneys precipitated at submarine hydrothermal springs, and these precipitates can be used successfully as flow-through reactors by feeding through multiple successive &#8220;hydrothermal&#8221; injections.

We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents.

Here we report experimental simulations of hydrothermal chimney growth using injection chemical garden methods. The versatility of this type of experiment ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Laboratory Production of Biofuels and Biochemicals from a Rapeseed Oil through Catalytic Cracking Conversion

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil (HGO) for production of transportation fuels through a fluid catalytic cracking (FCC) route. Cracking experiments are performed with a fully automated reaction unit at a fixed weight hourly space velocity (WHSV) of 8 hr-1, 490-530 &#176;C, and catalyst/oil ratios of 4-12 g/g. When a feed is in contact with catalyst in the fluid-bed reactor, cracking takes place generating gaseous, liquid, and solid products. The vapor produced is condensed and collected in a liquid receiver at -15 &#176;C. The non-condensable effluent is first directed to a vessel and is sent, after homogenization, to an on-line gas chromatograph (GC) for refinery gas analysis. The coke deposited on the catalyst is determined in situ by burning the spent catalyst in air at high temperatures. Levels of CO2 are measured quantitatively via an infrared (IR) cell, and are converted to coke yield. Liquid samples in the receivers are analyzed by GC for simulated distillation to determine the amounts in different boiling ranges, i.e., IBP-221 &#176;C (gasoline), 221-343 &#176;C (light cycle oil), and 343 &#176;C+ (heavy cycle oil). Cracking of a feed containing canola oil generates water, which appears at the bottom of a liquid receiver and on its inner wall. Recovery of water on the wall is achieved through washing with methanol followed by Karl Fischer titration for water content. Basic results reported include conversion (the portion of the feed converted to gas and liquid product with a boiling point below 221 &#176;C, coke, and water, if present) and yields of dry gas (H2-C2's, CO, and CO2), liquefied petroleum gas (C3-C4), gasoline, light cycle oil, heavy cycle oil, coke, and water, if present.

This paper presents an experimental method to produce biofuels and biochemicals from canola oil mixed with a fossil-based feed in the presence of a catalyst at mild temperatures. Gaseous, liquid, and solid products from a reaction unit are quantified and characterized. Conversion and individual product yields are calculated and reported.

The work is based on a reported study which investigates the processability of canola oil (bio-feed) in the presence of bitumen-derived heavy gas oil ...

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 ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

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. ...