This work was supported by National Institute of General Medical Sciences Grant R01 GM125943, National Geographic Grant CP-044R-17; Icelandic Research Fund Grant 152336-051; and University of Illinois at Chicago startup funds. Also, we thank the following contributors: Dr. Amanda Bulman for assistance with MALDI-TOF MS protein acquisition parameters; Dr. Terry Moore and Dr. Atul Jain for recrystallizing alpha-cyano-4-hydroxycinnamic acid matrix (CHCA).

<ol>
	<li>Sandrin, T. R., Goldstein, J. E., Schumaker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+TOF+MS+profiling+of+bacteria+at+the+strain+level:+A+review.">MALDI TOF MS profiling of bacteria at the strain level: A review.</a> Mass Spectrometry Reviews. 32 (3), 188-217 (2013).</li><li>Cain, T. C., Lubman, D. M., Weber, W. J., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+bacteria+using+protein+profiles+from+matrix-assisted+laser+desorption/ionization+time-of-flight+mass+spectrometry.">Differentiation of bacteria using protein profiles from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 8 (12), 1026-1030 (1994).</li><li>Holland, R. D., Wilkes, J. G., 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=Rapid+identification+of+intact+whole+bacteria+based+on+spectral+patterns+using+matrix-assisted+laser+desorption/ionization+with+time-of-flight+mass+spectrometry.">Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 10 (10), 1227-1232 (1996).</li><li>Rahi, P., Prakash, O., Shouche, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+time-of-flight+mass-spectrometry+(MALDI-TOF+MS)+based+microbial+identifications:+challenges+and+scopes+for+microbial+ecologists.">Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based microbial identifications: challenges and scopes for microbial ecologists.</a> Frontiers in Microbiology. 7, 1359 (2016).</li><li>Silva, R., Lopes, N. P., Silva, D. B. <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+MALDI+mass+spectrometry+in+natural+products+analysis.">Application of MALDI mass spectrometry in natural products analysis.</a> Planta Medica. 82, 671-689 (2016).</li><li>Clark, C. M., Costa, M. S., Sanchez, L. M., Murphy, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+MALDI-TOF+mass+spectrometry+protein+and+specialized+metabolite+analyses+to+rapidly+discriminate+bacterial+function.">Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (19), 4981-4986 (2018).</li><li>Freiwald, A., Sauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+classification+and+identification+of+bacteria+by+mass+spectrometry.">Phylogenetic classification and identification of bacteria by mass spectrometry.</a> Nature Protocols. 4 (5), 732-742 (2009).</li><li>Schulthess, B., Bloemberg, G. V., Zbinden, R., B&#246;ttger, E. C., Hombach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Bruker+MALDI+Biotyper+for+identification+of+Gram-positive+rods:+development+of+a+diagnostic+algorithm+for+the+clinical+laboratory.">Evaluation of the Bruker MALDI Biotyper for identification of Gram-positive rods: development of a diagnostic algorithm for the clinical laboratory.</a> Journal of Clinical Microbiology. 52 (4), 1089-1097 (2014).</li><li>Schumann, P., Maier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry+applied+to+classification+and+identification+of+bacteria.">MALDI-TOF mass spectrometry applied to classification and identification of bacteria.</a> Methods in Microbiology. 41, 275-306 (2014).</li><li>Chambers, M. C., Maclean, B., 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=A+cross-platform+toolkit+for+mass+spectrometry+and+proteomics.">A cross-platform toolkit for mass spectrometry and proteomics.</a> Nature Biotechnology. 30 (10), 918-920 (2012).</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 (2008).</li><li>Martens, L., Chambers, 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=mzML-a+community+standard+for+mass+spectrometry+data.">mzML-a community standard for mass spectrometry data.</a> Molecular &#38; Cellular Proteomics. 10 (1), (2011).</li></ol>

The authors have nothing to disclose.

The IDBac protocol details bacterial protein and specialized metabolite data acquisition and analysis of up to 384 bacterial isolates in 4 h by a single researcher. With IDBac there is no need to extract DNA from bacterial isolates or generate specialized metabolite extracts from liquid fermentation broths and analyze them using chromatographic methods. Instead, protein and specialized metabolite data are gathered by simply spreading material from bacterial colonies directly onto a MALDI target plate. This greatly reduces the time and cost associated with alternative techniques such as 16S rRNA gene sequencing and LCMS9.
It is important to add a matrix blank and calibration spots to the MALDI plate, and we recommend using an appropriate number of replicates to ensure reproducibility and statistical confidence. The numbers of replicates will be experiment-dependent. For example, if a user intends to differentiate thousands of colonies from a collection of environmental diversity plates, fewer replicates may be necessary (our lab collects three technical replicates per colony). Alternatively, if a user wishes to create a custom database of strains from specific bacterial taxa to rapidly determine sub-species classifications of unknown isolates, then more replicates are appropriate (our lab collects eight biological replicates per strain).
IDBac is a tool for rapidly differentiating highly-related bacterial isolates based on putative taxonomic information and specialized metabolite production. It can complement or serve as a precursor to orthogonal methods such as in-depth genetic analyses, studies involving metabolite production and function, or characterization of specialized metabolite structure by Nuclear Magnetic Resonance spectroscopy and/or LC-MS/MS.
Specialized metabolite production (IDBac MANs) is highly susceptible to bacterial growth conditions, especially using different media, which is a potential limitation of the method. However these traits may be exploited by the user, as IDBac can readily generate MANs showing the differences in specialized metabolite production under a variety of growth conditions. It is important to note that while specialized metabolite fingerprints may vary by growth condition, we have previously shown that protein fingerprints remain relatively stable across these variables (see Clark et al.6). When dealing with environmental diversity plates, we recommend purifying bacterial isolates prior to analysis in order to reduce possible contributions from neighboring bacterial cross-talk.
Finally, the lack of a searchable public database of protein MS fingerprints is a major shortcoming in the use of this method to classify unknown environmental bacteria. We created IDBac with this in mind, and included automated conversion of data into a community-accepted open-source format (mzML)10,11,12 and designed the software to allow searching, sharing, and creation of custom databases. We are in the process of creating a large public database (&#62;10,000 fully characterized strains), which will allow for the classification of some isolates to the species-level, including links to GenBank accession numbers when available.
IDBac is open source and the code is available for anyone to customize their data analysis and visualization needs. We recommend that users consult an extensive body of literature (Sauer et al.7, Silva et al.5) to help support and design their experimental goals. We host a forum for discussion at: https://groups.google.com/forum/#!forum/idbac and a means to report issues with the software at: https://github.com/chasemc/IDBacApp/issues.

A major barrier to researchers who study bacterial function is the ability to quickly and simultaneously assess the taxonomic identity of a microorganism and its capacity to produce specialized metabolites. This has prevented significant advances in understanding the relationship between bacterial phylogeny and specialized metabolite production in the majority of bacteria isolated from the environment. Although MS-based methods that use protein fingerprints to group and identify bacteria are well described1,2,3,4, these studies have generally been performed on small groups of isolates, in a species-specific manner. Importantly, information on specialized metabolite production, a major driver of microbial function in the environment, has remained unincorporated in these studies. Silva et al.5 recently provided a comprehensive history detailing the underuse of MALDI-TOF MS to analyze specialized metabolites and the shortage of software to relieve current bioinformatics bottlenecks. In order to address these shortcomings, we created IDBac, a bioinformatics pipeline that integrates both linear and reflectron modes of MALDI-TOF MS6. This allows users to rapidly visualize and differentiate bacterial isolates based on both protein and specialized metabolite MS fingerprints, respectively.
IDBac is cost-effective, high-throughput, and designed for the lay user. It is freely available (chasemc.github.io/IDBac), and only requires access to a MALDI-TOF mass spectrometer (reflectron mode will be required for specialized metabolite analysis). Sample preparation relies on the simple &#8220;extended direct transfer&#8221; method7,8 and data are collected with consecutive linear and reflectron acquisitions on a single MALDI-target spot. With IDBac, it is possible to analyze the putative phylogeny and specialized metabolite production of hundreds of colonies in under four hours, including sample preparation, data acquisition, and data visualization. This presents a significant time and cost advantage over traditional methods of identifying bacteria (such as gene sequencing), and analyzing metabolic output (liquid chromatography-mass spectrometry [LCMS] and similar chromatographic methods).
Using data obtained in linear mode analysis, IDBac employs hierarchical clustering to represent the relatedness of protein spectra. Since the spectra mostly represent ionized ribosomal proteins, they provide a representation of the phylogenetic diversity present in a sample. In addition, IDBac incorporates reflectron mode data to display specialized metabolite fingerprints as Metabolite Association Networks (MANs). MANs are bipartite networks that allow for easy visualization of shared and unique metabolite production between bacterial isolates. The IDBac platform allows researchers to analyze both protein and specialized metabolite data in tandem but also individually if only one data-type is acquired. Importantly, IDBac processes raw data from Bruker and Xiamen instruments, as well as txt, tab, csv, mzXML, and mzML. This eliminates the need for manual conversion and formatting of data sets, and significantly reduces the risk of user error or mishandling of MS data.&#160;

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>60-002-65</td>
			<td>LC-MS Ultra CHROMASOLV</td>
		</tr>
		<tr>
			<td>Autoflex Speed LEF MALDI-TOF instrument</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker Daltonics Bacterial test standard</td>
			<td>Fisher</td>
			<td>NC0884024</td>
			<td>Bruker Daltonics 8604530</td>
		</tr>
		<tr>
			<td>Bruker Peptide Calibration standard</td>
			<td>Fisher</td>
			<td>NC9846988</td>
			<td>Bruker Daltonics 8206195</td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Fisher Chemical</td>
			<td>A117-50</td>
			<td>99.5+%, Optima LC/MS Grade</td>
		</tr>
		<tr>
			<td>MALDI-TOF target Plate</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Chemical</td>
			<td>A456-500</td>
			<td>Optima LC/MS Grade</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>any is ok</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Fisher</td>
			<td>AC293810010</td>
			<td>99.5%, for biochemistry, ACROS Organics</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>VWR</td>
			<td>7732-18-5</td>
			<td>LC-MS</td>
		</tr>
		<tr>
			<td>&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td>Sigma</td>
			<td>28166-41-8</td>
			<td>(C2020-25G) &#8805;98% (TLC), powder</td>
		</tr>
	</tbody>
</table>

using the open-source maldi tof-ms idbac pipeline for analysis of microbial protein and specialized metabolite data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

We analyzed six strains of Micromonospora chokoriensis and two strains of Bacillus subtilis, which were previously characterized6, using data available at DOI: 10.5281/zenodo.2574096. Following directions in the Starting with Raw Data tab, we selected the option Click here to convert Bruker files and followed the IDBac-provided instructions for each dataset (Figure 14).
After the automated conversion and preprocessing/peak-peaking steps were completed, we proceeded to create a new combined IDBac experiment by transferring samples from the two experiments into a single experiment containing both Bacillus and Micromonosopora samples (Figure 15). The resulting analysis involved comparing protein spectra using mirror plots, as pictured in Figure 16, which was useful for evaluating spectra quality and adjusting peak-picking settings. Figure 17 displays a screenshot of the protein clustering results with default settings selected. The dendrogram was colored by adjusting the threshold on the plot (appears as a dotted line). Of note is the clear separation between genera, with both M. chokoriensis and B. subtilis isolates clustering separately.
Figure 18, Figure 19, and Figure 20 highlight the ability to generate MANs of user-selected regions by clicking and dragging across the protein dendrogram. With this we were able to rapidly create MANs to compare only the B. subtilis strains (Figure 18), only the M. chokoriensis strains (Figure 19), and all the strains simultaneously (Figure 20). The primary function of these networks is to provide researchers with a broad overview of the degree of specialized metabolite overlap between bacteria. With these data in hand, researchers now have the capacity to make informed decisions from only a small amount of material scraped from a bacterial colony.
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/59219/59219fig14v2.jpg" /> 
Figure 14: Spectra processing. Downloaded Bruker autoFlex spectra were converted and processed using IDBac. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig14v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/59219/59219fig15v2.jpg" /> 
Figure 15: Combined IDBac experiment. Because the Micromonospora and Bacillus spectra were collected on different MALDI target plates, the two experiments were subsequently combined into a single experiment-&#8220;Bacillus_Micromonsopora&#8221;. This was done within the &#8220;Work with Previous Experiments&#8221; tab, following directions within the menu &#8220;Transfer samples from previous experiments to new/other experiments&#8221;. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig15v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/59219/59219fig16v2.jpg" /> 
Figure 16: Comparison. Micromonspora and Bacillus spectra were compared using the mirror plots within the &#8220;Protein Data Analysis&#8221; page. Ultimately, default peak settings were chosen. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig16v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/59219/59219fig17v2.jpg" /> 
Figure 17: Hierarchical clustering. Hierarchical clustering, using default settings, correctly grouped Bacillus and Micromonospora isolates. The dendrogram was colored by &#8220;cutting&#8221; the dendrogram at an arbitrary height (displayed as a dashed-line) and 100 bootstraps used to show confidence in branching. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig17v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 18" class="xfigimg" src="/files/ftp_upload/59219/59219fig18v2.jpg" /> 
Figure 18: MAN created by selecting the Bacillus sp. strains from the protein dendrogram showed differential production of specialized metabolites. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig18v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 19" class="xfigimg" src="/files/ftp_upload/59219/59219fig19v2.jpg" /> 
Figure 19: MAN created by selecting the six Micromonospora sp. strains from the protein dendrogram showed differential production of specialized metabolites. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig19v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 20" class="xfigimg" src="/files/ftp_upload/59219/59219fig20v2.jpg" /> 
Figure 20: MAN of Bacillus sp. and Micromonospora sp. strains showing a differential production of specialized metabolites. <a href="https://www.jove.com/files/ftp_upload/59219/59219fig20v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data at JoVE.com

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

using-open-source-maldi-tof-ms-idbac-pipeline-for-analysis-microbial

Research

JoVE Journal

Biochemistry

19.0K Views.  University of Illinois at Chicago. IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

Video: Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, ...

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...

A Python-Based Tool for Accurate Mass Calculation of Biotherapeutic Entities

An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides an easy analytical tool for different stages of biopharmaceutical development. The protein's identity is confirmed when the experimental mass from MS is within a pre-defined mass error range of the theoretical mass. While several computational tools exist for the calculation of protein and peptide molecular weights, they either were not designed for direct application to biotherapeutic entities, have access limitations due to paid licenses, or require uploading protein sequences to host servers. 
We have developed a modular mass calculation routine that enables the easy determination of the average or monoisotopic masses and elemental compositions of therapeutic glycoproteins, including monoclonal antibodies (mAb), bispecific antibodies (bsAb), and antibody-drug conjugates (ADC). The modular nature of this Python-based calculation framework will allow the extension of this platform to other modalities such as vaccines, fusion proteins, and oligonucleotides in the future, and this framework could also be useful for the interrogation of top-down mass spectrometry data. By creating an open-source standalone desktop application with a graphical user interface (GUI), we hope to overcome the restrictions around use in environments where proprietary information cannot be uploaded to web-based tools. This article describes the algorithms and application of this tool, mAbScale, to different antibody-based therapeutic modalities.

Author Spotlight: Advancing Biotherapeutic Mass Calculation by Introducing mAbScale, a Python-Based Desktop Application

This article describes the use of a software application, mAbScale, for the calculation of masses for monoclonal antibody-based protein therapeutics.

Biotherapeutic masses are a means of verifying identity and structural integrity. Mass spectrometry (MS) of intact proteins or protein subunits provides ...