We gratefully thank Prof. Colin Jackson for the support with purified cofactor F420. This research was supported by Tyrolean science fund (TWF) and the Universit&#228;t Innsbruck (Publikationsfonds). We greatly acknowledge the support of GPS, HK, SB, GG, and HB.

NOTE: Extraction and analysis of cofactor F420 is a three-step process including sample lysis, cofactor pre-purification via solid-phase extraction (SPE), and cofactor detection via ion-paired-RP-HPLC (IP-RP-HPLC) with fluorescence detection. Prior to starting, prepare the materials and reagents as stated in Table 1.
1. Sample lysis
<ol>
	<li>Add up to 5 g of sample to appropriate tubes (e.g., 50 mL conical tubes).</li>
	<li>Add 5 mL of the lysis buffer (2x stock solution, Table 1) to the samples.</li>
	<li>Bring to a final volume of 10 mL with distilled water to reach a final concentration of 0.5 g&#183;mL-1.</li>
	<li>Vortex the diluted samples for 20 s.</li>
	<li>Autoclave for 30 min at 121 &#176;C.</li>
	<li>For dry samples like forest soil, bring to a final volume of 20 mL with distilled water after autoclaving and vortex the diluted sample. 
	&#8203;CAUTION: Temperature increase during autoclaving might cause tubes to burst.</li>
</ol>
2. Pre-purification of the cofactor F 420 via solid-phase extraction (SPE)
NOTE: All steps of SPE are conducted at room temperature
<ol>
	<li>Cool down the samples to room temperature.</li>
	<li>Centrifuge the autoclaved samples for 5 min at 11,000 x g.</li>
	<li>Prepare 5 mL SPE columns filled with 100 mg of weak anion mixed-mode polymeric sorbent.</li>
	<li>Condition the anion exchanger with 3 mL of methanol (condition solution, Table 1).</li>
	<li>Equilibrate the anion exchanger with 3 mL of distilled water (equilibration solution, Table 1).</li>
	<li>Load up to 9.0 mL of the supernatant from the centrifuged lysate onto the SPE column.</li>
	<li>Wash away impurities with 5 mL of 25 mM ammonium acetate (SPE wash solution 1, Table 1).</li>
	<li>Wash away&#160;impurities with 5 mL of methanol (SPE wash solution 2, Table 1).</li>
	<li>Elute the cofactor F420 in 1.0 mL of elution buffer (Table 1). 
	NOTE: Prepare fresh elution buffer. Due to the applied vacuum and the high vapor pressure of the elution buffer, elution volumes might differ from sample to sample. In order to secure the same final volume in all the samples, it is recommended to weigh the collection vessels before and after elution and calculate the effective elution volume. Balance the differences by the addition of elution buffer.</li>
</ol>
3. Detection of the cofactor F420
<ol>
	<li>Set the oven to 40 &#176;C and fluorescence detector to 420 nm extinction-wavelength and 470 nm emission-wavelength. Achieve separation via gradient mode using mobile phases A and B (Table 1): 0-3 min: 26% B; 3-24 min: 26%-50% B; 24-25 min: 50% B; 25-27 min: 50%-26% B; 27-35 min: 26% B at a flow rate of 0.75 mL&#183;min-1. 
	NOTE: Guarantee equilibration of column conditions prior to injecting samples by flushing the column at least with 3 column volumes of 74% mobile phase A and 26% mobile phase B (Table 1).
	<ol>
		<li>Filter the eluted samples from the SPE into HPLC vials using a PTFE filter with a pore size of 0.22 &#181;m. 
		NOTE: PTFE filters with a pore size of 0.22 &#181;m are recommended.</li>
		<li>Inject 50 &#181;L of the eluted sample onto the HPLC system to analyze the cofactor F420 composition and concentration. 
		NOTE: As no quantitative standard is used in this protocol, samples and variants have to be compared by peak area.</li>
	</ol>
	</li>
</ol>

<ol><li>Greening, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiology%2C+biochemistry%2C+and+applications+of+F420-+and+Fo-dependent+redox+reactions%5BTitle%5D)+AND+%22Microbiology+and+Molecular+Biology+Reviews%3A+MMBR%22%5BJournal%5D)">Physiology, biochemistry, and applications of F420- and Fo-dependent redox reactions.</a> Microbiology and Molecular Biology Reviews: MMBR. 80 (2), 451-493 (2016).</li>
<li>Bashiri, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+revised+biosynthetic+pathway+for+the+cofactor+F420+in+prokaryotes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A revised biosynthetic pathway for the cofactor F420 in prokaryotes.</a> Nature Communications. 10 (1), 451(2019).</li>
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<li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proposed+structure+for+coenzyme+F+420+from+methanobacterium%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Proposed structure for coenzyme F 420 from methanobacterium.</a> Biochemistry. 17 (22), 4583-4593 (1978).</li>
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<li>Eirich, L. D., Vogels, G. D., Wolfe, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+of+coenzyme+F420+and+properties+of+its+hydrolytic+fragments%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Distribution of coenzyme F420 and properties of its hydrolytic fragments.</a> Journal of Bacteriology. 140 (1), 20-27 (1979).</li>
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<li>Purwantini, E., Gillis, T. P., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+F420-dependent+glucose-6-phosphate+dehydrogenase+in+Mycobacterium+and+Nocardia+species%2C+but+absence+from+Streptomyces+and+Corynebacterium+species+and+methanogenic+Archaea%5BTitle%5D)+AND+%22FEMS+Microbiology+Letters%22%5BJournal%5D)">Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic Archaea.</a> FEMS Microbiology Letters. 146 (1), 129-134 (1997).</li>
<li>Purwantini, E., Daniels, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+a+novel+coenzyme+F420-dependent+glucose-6-phosphate+dehydrogenase+from+Mycobacterium+smegmatis%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.</a> Journal of Bacteriology. 178 (10), 2861-2866 (1996).</li>
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<li>Coats, J. H., Li, G. P., Kuo, M. -S. T., Yurek, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery%2C+production%2C+and+biological+assay+of+an+unusual+flavenoid+cofactor+involved+in+lincomycin+biosynthesis%5BTitle%5D)+AND+%22The+Journal+of+Antibiotics%22%5BJournal%5D)">Discovery, production, and biological assay of an unusual flavenoid cofactor involved in lincomycin biosynthesis.</a> The Journal of Antibiotics. 42 (3), 472-474 (1989).</li>
<li>Bown, L., Altowairish, M. S., Fyans, J. K., Bignell, D. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+the+Streptomyces+scabies+coronafacoyl+phytotoxins+involves+a+novel+biosynthetic+pathway+with+an+F420+-dependent+oxidoreductase+and+a+short-chain+dehydrogenase%2Freductase%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.</a> Molecular Microbiology. 101 (1), 122-135 (2016).</li>
<li>Gurumurthy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+F%28420%29+-dependent+anti-oxidant+mechanism+protects+Mycobacterium+tuberculosis+against+oxidative+stress+and+bactericidal+agents%5BTitle%5D)+AND+%22Molecular+microbiology%22%5BJournal%5D)">A novel F(420) -dependent anti-oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents.</a> Molecular microbiology. 87 (4), 744-755 (2013).</li>
<li>Greening, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mycobacterial+F420H2-dependent+reductases+promiscuously+reduce+diverse+compounds+through+a+common+mechanism%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Mycobacterial F420H2-dependent reductases promiscuously reduce diverse compounds through a common mechanism.</a> Frontiers in Microbiology. 8, 1000(2017).</li>
<li>Mathew, S., Trajkovic, M., Kumar, H., Nguyen, Q. -T., Fraaije, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enantio-+and+regioselective+ene-reductions+using+F420H2-dependent+enzymes%5BTitle%5D)+AND+%22Chemical+Communications%22%5BJournal%5D)">Enantio- and regioselective ene-reductions using F420H2-dependent enzymes.</a> Chemical Communications. 54 (79), Cambridge, England. 11208-11211 (2018).</li>
<li>Ney, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cofactor+tail+length+modulates+catalysis+of+bacterial+F420-dependent+oxidoreductases%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Cofactor tail length modulates catalysis of bacterial F420-dependent oxidoreductases.</a> Frontiers in Microbiology. 8, 1902(2017).</li>
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<li>Wagner, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medium+preparation+for+the+cultivation+of+microorganisms+under+strictly+anaerobic%2Fanoxic+conditions%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Medium preparation for the cultivation of microorganisms under strictly anaerobic/anoxic conditions.</a> Journal of Visualized Experiments: JoVE. (150), e60155(2019).</li>
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</ol>

The authors have nothing to disclose.

For the evaluation of cofactor F420 from methanogenic pure cultures, a microscopic evaluation can be performed to visualize the growth and activity (fluorescence microscopy) of the involved microorganisms (Figure 1). For samples deriving from natural environments, the use of microscopy to detect or quantify F420 is limited due to interferences with other fluorescent microorganisms, organic and inorganic particles. In this context, extraction of F420 and subse...

F420 is a widespread but often neglected cofactor, which functions as an obligate two-electron hydride carrier in primary and secondary metabolic processes of both Archaea and Bacteria1,2. F420 is a 5-deazaflavin and structurally similar to flavins, whereby its chemical and biological properties are more comparable with those of NAD+ or NADP+. Due to the substitution of nitrogen with carbon at position 5 of the isoalloxazine ring, it is a strong reductant, thus exhibiting a low standard redox potential of -340 mV1,3. F420 comprises a 5-deazaflavin ring and a 2-phospho-L-lactate linker (F420-0). An oligoglutamate tail containing n+1 glutamate monomers can be attached to the molecule (F420-n+1)4.
For a long time, the cofactor F420 has been solely associated with Archaea and Actinobacteria. This has largely been overturned. Recent analyses revealed that F420 is distributed among diverse anaerobic and aerobic organisms of the phyla Proteobacteria, Chloroflexi, and potentially Firmicutes inhabiting a myriad of habitats like soils, lakes, and the human gut1,5. In 2019, Braga et al.6, showed that the proteobacterium Paraburkholderia rhizoxinica produces a unique F420 derivative, containing a 3-phosphoglycerate instead of a 2-phospholactate tail, which might be widespread in various habitats. Within the domain Archaea, F420 has been found in several lineages, including methanogenic7, methanotrophic8,9, and sulfate-reducing orders10, and is supposed to be produced in Thaumarchaeota11.&#160;F420 is best known as an essential redox coenzyme in hydrogenotrophic and methylotrophic methanogenesis. The reduced form of F420 (F420H2) functions as an electron donor for the reduction of methylenetetrahydromethanopterin (methylene-H4MPT, Mer) and methenyl-H4MPT12,13. It can also be used as an electron carrier in H2-independent electron transport pathways of methanogens containing cytochromes12,14. Moreover, the oxidized form of F420 has a characteristic blue-green fluorescence upon excitation at 420 nm, which facilitates the detection of methanogens microscopically (Figure 1). Due to its low redox potential, F420 facilitates (i) the exogenous reduction of a broad spectrum of otherwise recalcitrant or toxic organic compounds, (ii) synthesis of tetracycline and lincosamide antibiotics or phytotoxins in streptomycetes (phylum Actinobacteria), and (iii) resistance to oxidative or nitrosative stress or other unfavorable conditions in mycobacteria (phylum Actinobacteria)1,5,15,16,17,18,19,20,21,22. Consequently, F420-dependent oxidoreductases are promising biocatalysts for industrial and pharmaceutical purposes as well as for bioremediation of contaminated environments1,23. Despite these recent findings, the exact roles of the cofactor F420 are still marginally known in Actinobacteria or other bacterial phyla.
There are at least three pathways for F420 biosynthesis2,6,24. In the beginning, the biosynthesis pathway is split into a 5-deazaflavin biosynthesis and a 2-phospholactate metabolism branch. The reactive part of the F420 molecule is synthesized via FO-synthase using the substrates tyrosine and 5-amino-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. The result is the riboflavin level chromophore FO. Within the currently accepted lactate metabolism branch, L-lactate is phosphorylated to 2-phospho-L-lactate by an L-lactate kinase (CofB); 2-phospho-L-lactate, in turn, is guanylated to L-lactyl-2-diphospho-5&#39;-guanosine by 2-phospho-L-lactate guanylyltransferase (CofC). In the next step, L-lactyl-2-diphospho-5&#39;-guanosine is linked to FO by a 2-phospho-L-lactate transferase (CofD) to form F420-02. Finally, the enzyme F420-0:&#611;-glutamyl ligase (CofE) ligates glutamate monomers to F420-0, forming the final cofactor6 in varying numbers23,25. Different organisms show different patterns in the number of attached glutamate residues, with shorter tails found for methanogens than in mycobacteria2,25,26. Generally, methanogens show tail lengths from two to three, with up to five in the acetoclastic methanogen, Methanosarcina sp., while tail lengths found in Mycobacterium sp. ranged from five to seven glutamate residues2,25,26,27. However, recent findings showed that long-chain F420 binds to F420-dependent oxidoreductases with a higher affinity than short-chain F420; moreover, bound long-chain F420 increases the substrate affinity but decreases the turnover rate of respective enzymes23.
Detection of cofactor F420 is often based on its fluorescence. Thereby, its oligo glutamate derivates were separated using reversed-phase (RP)-HPLC27,28. Recently, Ney et al. used tetrabutylammonium hydroxide as an ion-pairing reagent for the negatively charged glutamate tail to enhance separation on RP-HLPC successfully5. Here, we present a method for the preparation of samples, subsequent lysis, extraction, purification, separation, and quantification of cofactor F420 not only from pure cultures but also from different environmental samples (i.e., soils and digester sludge).

<table><tbody><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>Biocompatible HPLC system equipped with gradient modul, oven and fluorescence detector</td><td>Shimadzu</td><td></td><td>HPLC system</td></tr><tr><td>Centrifuge and rotor for 50 mL &amp;ldquo;Falcon&amp;rdquo; tubes (11.000 rcf) and appropriate tubes</td><td></td><td></td><td></td></tr><tr><td>HPLC Column: Gemini NX C18, 5 &amp;mu;m, 150 x 3 mm</td><td>Phenomenex</td><td></td><td>HPLC column</td></tr><tr><td>PTFE filter (pore size 0.22 &amp;micro;m) to remove particulate matter prior HPLC analysis</td><td></td><td></td><td></td></tr><tr><td>Resin for SPE: Strata-X-AW 33 &amp;mu;m as weak anion mixed-mode polymeric sorbent</td><td>Phenomenex</td><td></td><td>weak anion mixed-mode polymeric sorbent</td></tr><tr><td>Vacuum manifold for SPE and appropriate collection tubes</td><td></td><td></td><td>SPE equipment</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr></tbody></table>

extraction of cofactor f420 for analysis of polyglutamate tail length from methanogenic pure cultures and environmental samples

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.

Pure cultures of Methanosarcina thermophila and Methanoculleus thermophilus, both thermophilic methanogenic Archaea, were grown in suitable media as described previously29,30. For Methanosarcina thermophila, methanol was used as an energy source, whereas Methanoculleus thermophilus was grown on H2/CO2. Growth was checked by microscopic evaluation, while activity was examined via measurement of met...

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Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

A method for the extraction of cofactor F420 from pure cultures was optimized for the liquid chromatographic separation and analysis of F420 tail length in pure culture and environmental samples.

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is ...

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Research

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2.6K Views.  Universität Innsbruck. A method for the extraction of cofactor F420 from pure cultures was optimized for the liquid chromatographic separation and analysis of F420 tail length in pure culture and environmental samples.

Video: Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Biochemistry

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Chemistry

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Biology

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

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

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

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

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

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

Monitoring GTPase Atlastin in Different Nucleotide Loading States

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule F&#246;rster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

Extraction of Cofactor F₄₂₀ for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples

Summary

Explore More Videos

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

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

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin