Name: toxin induction and protein extraction from fusarium spp. cultures for proteomic studies
Uploaded: 2010-02-16T15:05:00
Duration: 8 min 19 s
Description: Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

We thank Servane Contal and Boris Untereiner for their technical contribution. We acknowledge the support of FNR "FUTOX" project.

The protocol requires 16 days for its completion. The timeframe is detailed in figure 1.
Details for buffer preparation
<ul>
 <li>Preparation of the lysis buffer (3mL per sample).</li>
 <li>Preparation of the washing buffer (50mL per sample). This buffer should be pre-chilled and stored at -20&deg;C in the freezer until further analysis and kept on ice during the entire procedure.</li>
 <li>Preparation of the precipitation buffer (20mL per sample). This buffer should be pre-chilled and stored at -20&deg;C in the freezer until needed and kept on ice during the entire procedure.</li>
 <li>The work should preferentially be carried out into the cold chamber at 4&deg;C. </li>
</ul>
Fungal species
Three strains used for proteomic analyses were classified to be Fusarium graminearum on the basis of their morphological features and Translation-Elongation factor Alpha-1 analysis (O'Donnell et al., 1998)
These strains belonged to the CRP-Gabriel Lippmann collection and were maintained and stored at - 80&deg;C in 15% glycerol. 
Preparation of mycelia
Fungi were cultivated on V8 for 4 days at 25&deg;C alternating light and dark period each 12 hrs. Cultures were fragmented using a sterile blade and pieces of these cultures were used to inoculate 250 ml Erlenmeyer flasks containing 100 mL of toxin-inducing media. Mycelia were harvested after 10 days and were separated from medium by filtering the culture with a sterile filter. Mycelia were washed 3 times with sterile water to remove medium residues and other compounds. We then immediately froze the cells by adding liquid nitrogen and kept the samples at -80&deg;C.
General description of the protein extraction
Protein samples were extracted using the method described in the figure 3A and 3B. The Fusarium samples were ground in liquid nitrogen and the powder was collected in 10 ml Teflon tubes. The samples were then incubated 30 minutes with the lysis buffer, boiled for 10 minutes, and centrifuged 2 times at room temperature (12000g for 15 minutes). The supernatants were collected and incubated at -20 &deg;C with precipitation buffer overnight. After centrifugation at 4&deg;C 30000g for 45 minutes, the pellets of the precipitated proteins were washed 3 times with cold acetone containing DTT. Finally, the pellets were air-dried and the proteins were resolubilized in the labelling buffer, adjusting pH to 8. A sub-sample of 30 ml was removed for total protein quantification and the remaining supernatant was stored at -20&deg;C until protein electrophoresis.
<img src="/files/ftp_upload/1690/1690fig1.jpg" alt="Figure 1" /> Figure 1. Timeline of the procedure.
<img src="/files/ftp_upload/1690/1690fig2.jpg" alt="Figure 2" /> Figure 2. Scheme for multiple operators sample-processing.
<img src="/files/ftp_upload/1690/1690fig3atmb.jpg" alt="Figure 3a" border="0" /> Figure 3a. <img src="/files/ftp_upload/1690/1690fig3btmb.jpg" alt="Figure 3b" border="0" /> Figure 3b. Please <a href="https://www.jove.com/files/ftp_upload/1690/1690fig3a.jpg">click here</a> to see a larger version of figure 3a, or <a href="https://www.jove.com/files/ftp_upload/1690/1690fig3b.jpg">here</a> for a larger version of figure 3b. 
Figures 3a-b. Flow charts for the protein extraction procedure (A: first day; B: second day).
<img src="/files/ftp_upload/1690/1690fig4tmb.jpg" alt="Figure 4" border="0" /> Figure 4. 1D picture of protein extracts. Proteins were run until complete migration of blue to the end of the gel that was stained with lava Purple. Please <a href="https://www.jove.com/files/ftp_upload/1690/1690fig4.jpg">click here</a> to see a larger version of figure 4.

<ol>
	<li>Bridge, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Extraction+from+Fungi.">Protein Extraction from Fungi.</a> Protein Purification Protocols. , 39-48 (1996).</li><li>Jiao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+carbon+sources+on+trichothecene+production+and+Tri+gene+expression+by+Fusarium+graminearum+in+liquid+culture.">Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture.</a> FEMS Microbiol Lett. 285, 212-219 (2008).</li><li>Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+of+filamentous+fungi.">Proteomics of filamentous fungi.</a> Trends in Biotechnology. 25, 395-400 (2007).</li><li>Milles, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+proteomic+approach+to+monitor+protein+synthesis+in+mycotoxin+producing+moulds.">Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.</a> Mycotoxin Res. 23, 161-165 (2007).</li><li>Morrison, N., Field, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concept+of+sample+in+OMICS+technology.">Concept of sample in OMICS technology.</a> OMICS. 10, 127-137 (2006).</li><li>O'Donnell, K., Kistler, H. C., Cigelnik, E., Ploetz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+evolutionary+origins+of+the+fungus+causing+Panama+disease+of+banana:+Concordant+evidence+from+nuclear+and+mitochondrial+gene+genealogies.">Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies.</a> PNAS. 95, 2044-2049 (1998).</li><li>Paper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+extracellular+proteins+in+vitro+and+in+planta+from+the+pathogenic+fungus+Fusarium+graminearum.">Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.</a> Proteomics. 7, 3171-3183 (2007).</li><li>Pasquali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video+in+science.+Protocol+videos:+the+implications+for+research+and+society.">Video in science. Protocol videos: the implications for research and society.</a> EMBO Rep. 8, 712-716 (2007).</li><li>Taylor, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+information+about+a+proteomics+experiment+(MIAPE).">The minimum information about a proteomics experiment (MIAPE).</a> Nat Biotechnol. 25, 887-893 (2007).</li><li>Taylor, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analyses+of+Fusarium+graminearum+grown+under+mycotoxin-inducing+conditions.">Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.</a> Proteomics. 8, 2256-2265 (2008).</li></ol>

Recent interest in proteomic approaches within the fungal biology domain led to an increased number of publications using this technique (as reviewed in Kim et al., 2007). Methods for protein extraction rely on a combination of extraction procedures; they are often scarcely described in the methodological sections and require expertise in dealing with the potential troubleshooting methods.
As for other "omics" approaches, setting standards for sample and data processing is essential in order to generate reliable scientific information. Moreover samples and procedures of manipulation should be adequately described in order to allow shared result interpretation among different "omics" experiments. This would be essential for fully exploiting potentiality of cross-data mining in genomics, proteomics, metabolomics, ... (Morrison et al., 2006). Minimum information about a proteomic experiment has been drafted and working groups have been established in order to tackle all the aspects of an experiment (Gel electrophoresis, Mass Spectrometry, Molecular Interactions, Protein Modifications, Proteomics Informatics, Sample Processing). At the moment no defined information are available on sample processing procedures. Given the importance of protein extraction procedures in determining the final quality of proteomic studies, in order to implement procedures that can increase standardization within the framework of MIAPE guidelines (Taylor et al., 2007), we proposed the use of a video protocol detailing sample processing. Video description of experiments may contribute substantially to increase the number of information on sample processing that may result in better reproducibility of "omics" experiments (Pasquali, 2007).
Indeed, reproducibility across laboratories is a fundamental requirement in order to guarantee validity of proteomics results (http://www.fixingproteomics.org). The cross-laboratory reproducibility is influenced by two factors: different instrumentations and different operators manipulating samples.
In the described protocol, we propose to perform biological replicates involving multiple operators (Figure 2 and video) to increase the reliability of data (i.e. technical variation of the results is taken into account).
The procedure described for protein extraction is based on SDS and heating. This was previously shown to guarantee a good purity and quantity of proteins (Bridge 1996). SDS coupled with boiling dissolves cell walls, hydrophobic proteins and prevents the formation of oligomers that abort precipitation of proteins. Boiling allows also inactivation of proteases. The same effect is produced also by EDTA, PMSF and complete mini protease inhibitor. DTT removes disulfide bridges in and between proteins facilitating protein solubilization.
In order to remove SDS before IEF, proteins are precipitated by acetone/TCA/DTT. Furthermore, acetone/TCA/DTT precipitation allows removing some contaminants such as lipids, nucleic acids, salts or/and phenolic compounds when these compounds are present. Like SDS, these molecules prevent a good migration during IEF. Following this precipitation, it is necessary to wash the precipitated proteins by acetone/DTT, in order to remove TCA as it can interfere with IEF.
A good sample preparation is the key to good results. For this, it is also essential to avoid contamination of proteins from the environment working with powder free gloves and respecting good laboratory practices. From a technical point of view, within the described protocol, the most critical steps for obtaining sufficient amounts and purity of proteins are two phases. First, the grinding phase where complete destruction of the cell is fundamental to release proteins; and second, the purification of total proteins, a phase encompassing the removal of the majority of lipids, DNA and other contaminants that may interfere with the migration of proteins.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
			<div>
			Media and solutions
PDA: 39 g Potato Dextrose Agar (Difco); 1 L distilled water. 
V8 agar:&nbsp; 200 ml V8 (campbell&rsquo;s, USA), 2g CaCO3 (Sigma), 16g Agar (DIFCO), 800 ml distilled water
Toxin inducing media: 1g K2HPO4, 0.5g KCl, 0.5g MgSO4 7H2O, 10 mg FeEDTA, 2g L glutamic acid, 10g sucrose in 1L of distilled water.
Lysis Buffer:
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>100mL</td>
 <td>Final concentration </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris-HCL pH8</td>
 <td>5mL</td>
 <td>50mM </td>
 <td>1M</td>
 </tr>
 <tr>
 <td>SDS</td>
 <td>2g or 10mL</td>
 <td>2%</td>
 <td>20%</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>500mL</td>
 <td>10mM</td>
 <td>2M</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>20mL</td>
 <td>0.1mM</td>
 <td>0.5M</td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>200mL</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Complete mini protease inhibitor</td>
 <td>1 tablet</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Water&nbsp;</td>
 <td>Qsp</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>
Precipitation Buffer:
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>100mL</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>TCA </td>
 <td>20mL</td>
 <td>20%</td>
 </tr>
 <tr>
 <td>DTT </td>
 <td>0.1mL</td>
 <td>0.1%</td>
 </tr>
 <tr>
 <td>Aceton</td>
 <td>Qsp</td>
 <td>&nbsp;</td>
 </tr>
</table>
Washing Buffer:
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>100mL</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>DTT </td>
 <td>0.1mL</td>
 <td>0.1%</td>
 </tr>
 <tr>
 <td>Aceton</td>
 <td>100mL</td>
 <td>&nbsp;</td>
 </tr>
</table>
Labelling Buffer (store at -18&deg;C in small aliquots):
<table border="1">
 <tr>
 <td>&nbsp;</td>
 <td>1mL</td>
 <td>Final concentration </td>
 </tr>
 <tr>
 <td>Urea </td>
 <td>140 mg</td>
 <td>7M </td>
 </tr>
 <tr>
 <td>Thiourea </td>
 <td>50 mg</td>
 <td>2M </td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>40 mg</td>
 <td>4% </td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>30 &micro;L</td>
 <td>30 mM </td>
 </tr>
 <tr>
 <td>Water</td>
 <td>1 mL</td>
 <td>&nbsp;</td>
 </tr>
</table>		</div>

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).

Watch this Scientific Journal Video about Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies at JoVE.com

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.

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, ...

toxin-induction-protein-extraction-from-fusarium-spp-cultures-for

Research

JoVE Journal

Biology

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...