This study was supported by an NSF Career Grant (MCB0954016) and a DOE Bioenergy Research Grant (DEFG0208ER64694).

1.	Cell culture (Figure 1)
<ol>
	<li>Grow cells in minimal medium with trace elements, salts, vitamins, and specifically labeled carbon substrates that are best for pathway investigation. Use either shaking flasks or bioreactors for cell culture. Organic nutrients, such as yeast extract, may interfere with the measurement of amino acid labeling and thus cannot be present in the culture medium.	</li>
	<li>Monitor cell growth by the optical density of the culture at an optimal wavelength (e.g., OD730 for Cyanothece 51142) with a UV/Vis spectrophotometer.</li>
	<li>Cells can first be grown in a non-labeled medium. The middle-log growth phase cells are preferred to be used for inoculation (3% (v/v) by volume inoculation ratio) of the labeled medium. The labeled culture should be sub-cultured (3% v/v inoculation ratio) in the same labeled medium to avoid the introduction of non-labeled carbon from the initial inoculum.</li>
</ol>
2.	Amino acid extraction
<ol>
	<li>Harvest sub-cultured cells (10mL) in the middle-log growth phase by centrifugation (10 min, 8000&times;g).</li>
	<li>Resuspend the pellet in 1.5mL of 6M HCl and transfer it to a clear glass, screw-top GC vial. Cap the vials and place them in a 100&deg;C oven for 24 hours to hydrolyze the biomass proteins into amino acids. Hydrolysis of biomass pellets can yield 16 of the 20 common amino acids (Figure 2) 5. Cysteine and tryptophan are degraded, and glutamine and asparagine are converted to glutamate and aspartate, respectively. </li>
	<li>Centrifuge the amino acid solution at 20,000&times;g for 5 min using 2 ml Eppendorf tubes, and transfer the supernatants to new GC vials. This step removes solid particles in the hydrolysis solution.</li>
	<li>Remove the GC vial lids and dry the samples completely under a stream of air using a Thermo Scientific Reacti-Vap evaporator (note: a freeze dryer can also be used to dry samples).&nbsp; This step can be done overnight.</li>
</ol>
3.	Amino acid derivatization and GC-MS conditions
Analysis of amino acids or charged/highly polar metabolites via GC requires that these metabolites be derivatized, so that the amino acids are volatile and can be separated by gas chromatography 2. 
<ol>
	<li>Dissolve the dried samples with 150 &mu;L of tetrahydrofuran (THF) and 150 &mu;L of N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide derivatization reagent.</li>
	<li>Incubate all samples in an oven or a water bath between 65 and 80&deg;C for 1 hour. Vortex occasionally to make sure the metabolites in the vial are dissolved.</li>
	<li>Centrifuge the samples at 20,000&times;g for 10 min, and then transfer the supernatant to new GC vials. The supernatant should be a clear and yellowish solution. Due to saturation of the detectors, GC-MS measurement accuracy can be affected by the high concentration of injected TBDMS derivatized amino acids (these samples often shows dark brown color), therefore, we should dilute these samples using THF before GC-MS measurement 6.</li>
	<li>Analyze the samples by GC-MS (use a 1:5 or 1:10 split ratio, injection volume = 1 &mu;L, carrier gas helium = 1.2 mL/min). Use the following GC temperature program: hold at 150&deg;C for 2 minutes, increase at 3&deg;C per min to 280&deg;C, increase at 20&deg;C per min to 300&deg;C, and then hold for 5 minutes. Solvent delay can be set as ~5 min (for a 30 meter GC column). The range of the mass to charge ratio (m/z) in MS can be set between 60 and 500. </li>
</ol>
4.	GC-MS data analysis
<ol>
	<li>TBDMS derivatized amino acid measurement can also be affected by isotope discrimination in GC separation. Light isotopes move slightly faster than heave isotopes in GC column. To reduce the potential measurement bias, we may average the mass spectrum of the whole amino acid peak range 6</li>
	<li>The GC and MS spectra of TBDMS derivatized metabolites have been reported before7. The GC retention time and the unique m/z peaks for each amino acid are illustrated in Figure 3.</li>
	<li>Derivatization of amino acids or central metabolites introduces significant amounts of naturally-labeled isotopes, including 13C (1.13%), 18O (0.20%), 29Si (4.70%), and 30Si (3.09%). The measurement noise from natural isotopes in the raw mass isotopomer spectrum can be corrected by using published software5, 8. The final isotopic labeling data are reported as mass fractions, e.g., M0, M1, M2, M3 and M4 (representing fragments containing zero to four 13C labeled carbons).</li>
	<li>Measurement of amino acids can provide isotopic labeling information about eight crucial precursor metabolites: 2-oxo-glutarate, 3-P-glycerate, acetyl-CoA, erythrose-4-P, oxaloacetate, phosphoenolpyruvate, pyruvate, and ribose-5-P. The labeling patterns in these metabolites can be used to identify several central metabolic pathways (Figure 2) 9. The outcome of the labeling experiments can be further confirmed using other biochemistry methods (e.g., RT-PCR).</li>
</ol>
5.	Pathway analysis using labeled amino acid data
By investigating only a few key amino acids produced from well-designed 13C tracer experiments, we may reveal several unique pathways or enzyme activities without performing sophisticated 13C-metabolic flux analysis of entire central metabolism.
<ol>
	<li>Entner&ndash;Doudoroff pathway: [1-13C] glucose can be used as the carbon source. If the pathway is active, serine labeling will be significantly lower than labeling in alanine 10.</li>
	<li>Branched TCA cycle: [1-13C] pyruvate can be used as the carbon source. If the TCA cycle is broken, aspartate can be labeled by two carbons, while glutamate is labeled with only one carbon 11, 12.</li>
	<li>CO2 fixation by Calvin-Benson-Bassham cycle in a mixotrophic metabolism: Non-labeled CO2 and labeled carbon substrates are both used as the carbon sources. If Calvin cycle is functional, serine and histidine labeling will be significantly diluted, comparing to other amino acids. Such method can determine the relative CO2 fixation when organic carbon sources are present in the medium 13.</li>
	<li>Oxidative pentose phosphate pathway: [1-13C] glucose can be used as the carbon source. If the pathway is active, non-labeled alanine will be &gt;50% 12.</li>
	<li>Anaplerotic pathway (e.g., PEP + CO2 &agrave; oxaloacetate): 13CO2 and non-labeled carbon substrates (e.g., glycerol or pyruvate) can be used as the carbon source. If the pathway is active, aspartate labeling will be significantly enriched, comparing to alanine and serine 13.</li>
	<li>Re-citrate synthase: [1-13C] pyruvate can be used as the carbon source. If the enzyme is active, glutamate is labeled in &beta;-carboxyl group 3, 4.</li>
	<li>Citramalate pathway: [1-13C] pyruvate, [2-13C] glycerol, or [1-13C] acetate can be used as the carbon source. If the pathway is active, leucine and isoleucine labeling amounts are identical 14. </li>
	<li>Serine-isocitrate lyase cycle: [1-13C] pyruvate or [1-13C] lactate can be used as the carbon source. If the pathway is active, the third position carbon in serine will be labeled 15.</li>
	<li>Utilization of nutrients (i.e., exogenous amino acids): a culture medium with fully labeled carbon substrates and non-labeled amino acids can be used. If the cells selectively utilize these supplemented non-labeled nutrients, we will see significant labeling dilution of these amino acids in the biomass. This method can be used to investigate which nutrient supplements are preferred by the cell 16.</li>
</ol>6. Representative Results
Recent bioenergy studies have revived interests in using novel phototrophic microorganisms for bioenergy production and CO2 capture. In the past years, quite a few 13C-metabolism analyses, including advanced 13C-Metabolic Flux Analyses (13C-MFA), have been applied to investigate central metabolisms in phototrophic bacteria, because biochemical knowledge of the central metabolic pathways is not well-founded in these non-model organisms10, 11, 17-20. Here, we present an example of the discovery of an alternate isoleucine pathway in Cyanothece 51142 21. Cyanothece 51142 does not contain the enzyme (EC 4.3.1.19, threonine ammonia-lyase), which catalyzes conversion of threonine to 2-ketobutyrate in the typical isoleucine synthesis pathway. To resolve the isoleucine pathway, we grow Cyanothece 51142 (20 mL) in ASP2 medium 22 with 54 mM glycerol (2-13C, &gt;98%). Cyanothece 51142 utilizes 2nd position labeled glycerol as the main carbon source. We observe that threonine and alanine have one labeled carbon, while isoleucine is labeled with three carbons. Therefore, synthesis in Cyanothece 51142 cannot be derived from the threonine route employed by most organisms (Figure 4). On the other hand, leucine and isoleucine have identical labeling patterns based on fragment (M-15)+ and fragment (M-159)+. For example, the isotopomer data from [M-15]+ (containing unfragmented amino acids) show identical labeling for leucine (M0=0.01, M1=0.03, M2=0.21, M3=0.69) and isoleucine (M0=0.01, M1=0.03, M2=0.24, M3=0.67). Thus leucine and isoleucine must be synthesized from the same precursors (i.e., pyruvate and acetyl-CoA). This observation is consistent with the labeled carbon transition in the citramalate pathway for isoleucine synthesis. To confirm this pathway, we search the Joint Genome Institute database and find the presence of a citramalate synthase CimA (cce_0248) in Cyanothece. 
<img src="/files/ftp_upload/3583/3583fig1.jpg" alt="Figure 1"/> Figure 1.&nbsp;
The 13C-assisted pathway analysis steps. 
<img src="/files/ftp_upload/3583/3583fig2.jpg" alt="Figure 2"/> Figure 2.&nbsp;
Amino acids used for acquiring the labeling pattern of their metabolic precursors. ACoA, acetyl-CoA; AKG, &alpha;-Ketoglutarate; C5P, ribose 5-phosphate; CIT, citrate; E4P, erythrose 4-phosphate; G6P, glucose 6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PGA, 3-phosphoglycerate; PYR, pyruvate.
<img src="/files/ftp_upload/3583/3583fig3.jpg" alt="Figure 3"/> Figure 3.&nbsp;GC peaks for 16 amino acids. TBDMS derivatized amino acids are cracked by MS into two fragments: (M-57)+, containing the entire amino acid, and (M-159)+, which lacks the &alpha; carboxyl group of the amino acid. For leucine and isoleucine, the (M-57)+ was overlapped by other mass peaks. We suggest using fragment (M-15)+ to analyze the entire amino acid labeling. The (f302)+ group is detected in most amino acids, which contains only the first (&alpha;-carboxyl group) and second carbons in an amino acid backbone. Because this MS peak often has high noise-to-signal ratios, (f302)+ is not recommended for quantitatively analyzing the metabolic fluxes7.
<img src="/files/ftp_upload/3583/3583fig4.jpg" alt="Figure 4"/> Figure 4.&nbsp;
Labeling transitions in isoleucine pathways in Cyanothece 51142 (modified from our previous paper)21.

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No conflicts of interest declared.

This protocol consists of feeding the cell with a labeled substrate and measuring the resulting isotopic labeling patterns in the amino acids via GC-MS. Since MS data (m/z ratios) give just the overall amount of labeling of MS ions, we have to assess the isotopomer distributions of amino acids by examining the m/z ratios of both unfragmented (M-57)+ and fragmented amino acids (i.e., (M-159)+ and (f302)+). Furthermore, we can perform several cell cultures with a chemically identical medium...

<table border="1">
<tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>TBDMS</td>
 <td>Sigma-Aldrich</td>
 <td>19915</td>
 <td>-</td>
 </tr>
 <tr>
 <td>THF</td>
 <td>Sigma-Aldrich</td>
 <td>34865</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Labeled carbon substrate</td>
 <td>Cambridge Isotope Laboratories</td>
 <td>Depend on the experimental requirement</td>
 <td>Website:
 <a href="http://www.isotope.com">http://www.isotope.com</a></td>
 </tr>
 <tr>
 <td>Gas chromatograph</td>
 <td>Agilent Technologies</td>
 <td>Hewlett-Packard, model 7890A</td>
 <td>-</td>
 </tr>
 <tr>
 <td>GC Columns</td>
 <td>J&amp;W Scientific, Folsom, CA</td>
 <td>DB5 (30m)</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Mass spectrometer</td>
 <td>Agilent Technologies</td>
 <td>5975C</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Reacti-Vap Evaporator</td>
 <td>Thermo Scientific</td>
 <td>TS-18825</td>
 <td>For drying amino acid samples</td>
 </tr>
</tbody>
</table>

metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

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Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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JoVE Journal

Biology

24.2K 조회수.  Washington University. 13 C - 동위 원소 라벨링은 미생물의 다양한 유형의 셀 중앙 신진 대사를 결정하기위한 유용한 기법입니다. 세포가 특정 레이블 기판과 교양되었습니다 후, GC - MS 측정은 proteinogenic 아미노산의 고유 라벨 패턴을 기반으로 기능성 대사 경로를 확인할 수 있습니다.

비디오: Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

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 ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.