Name: An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis (Video) | JoVE Journal
Uploaded: 2015-09-15T14:00:00.000+00:00
Duration: 8 min 9 s
Description: Learn an aquatic microbial metaproteomics workflow for extracting proteins and DNA from seawater samples. This method generates tryptic peptides suitable for tandem mass spectrometry analysis.

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5 minutes

The authors would like to acknowledge Marcos Di Falco for his expertise and advice with the preparation of the samples for nano-LC MS/MS as well as Dr. Zoran Minic from the University of Regina for the LC MS/MS analysis. This work was supported by NSERC (DG402214-2011) and CRC (950-221184) funding. D.C. was supported by Concordia Institute for Water, Energy, and Sustainable Systems and FQRNT.

1. Prepare Reagents
<ol>
	<li>Prepare SDS-extraction solution: 0.1 M Tris-HCl pH 7.5, 5 % glycerol, 10 mM EDTA and 1 % SDS. Filter-sterilize using a 0.22 &#181;m filter and store at 4 &#176;C.</li>
	<li>Prepare stock reagents needed for the polyacrylamide gel.
	<ol>
		<li>Prepare 1.5 M Tris-HCL pH 8.8. Filter-sterilize using a 0.22 &#181;m filter and store at room temperature.</li>
		<li>0.5 M Tris-HCL pH 6.8. Filter-sterilize using a 0.22 &#181;m filter and store at room temperature.</li>
		<li>Prepare 10 % SDS. Filter sterilize using a 0.22 &#181;m filter and store at room temperature.</li>
	</ol>
	</li>
	<li>Prepare solutions needed for in-gel trypsin digest and peptide extraction.
	<ol>
		<li>Prepare 100 mM ammonium bicarbonate. Filter-sterilize using a 0.22 &#181;m filter and store at 4 &#176;C.</li>
		<li>Prepare 1 M DTT stocks. Suspended in 100 mM ammonium bicarbonate and store at -80 &#176;C.</li>
		<li>Prepare 550 mM iodoacetamide stocks. Suspended in 100 mM ammonium bicarbonate and store at -80 &#176;C.</li>
		<li>Prepare 100 ng/&#181;l trypsin stocks. Aliquot 60 &#181;l into 1.5 ml microcentrifuge tubes and store at -80 &#176;C.</li>
		<li>Prepare extraction solution: 1% formic acid, 2% acetonitrile.</li>
		<li>Prepare resuspension solution: 5% acetonitrile and 0.1% formic acid.</li>
	</ol>
	</li>
</ol>
2. Perform Cell Lysis in Cartridge Filter Unit with Cells Preserved in RNA Stabilization Solution
<ol>
	<li>Expel the RNA stabilization solution from the cartridge filter unit (1.5 ml) and into a 2 ml microcentrifuge tube using a 60 ml syringe attached at the luer-lock end of the filter.</li>
	<li>Centrifuge the 2 ml microcentrifuge tube for 10 min&#160;at 17,000 x g to pellet any cellular debris. This is done so that the filter in step 2.3 does not clog.</li>
	<li>Transfer supernatant to a 10 K ultracentrifugal filter unit to capture any proteins originating from cells that have lysed from the freeze/thaw of the sample. Do not discard the pellet. It will be used in step 2.5. Perform centrifugation of the ultracentrifugal filter unit at 3,270 x g for 30 min&#160;or until the volume has reduced to about 600 &#181;l.</li>
	<li>Melt the tip of a p10 tip and block the non luer-lock side of the cartridge filter unit with the open end of the tip. This will ensure that no extraction buffer and biomass escapes during steps 2.5-2.7.</li>
	<li>Suspend the pellet in 1 ml SDS-extraction solution and pipette it into the original cartridge filter unit. The easiest way to pipette into the cartridge filter unit is to stack a p200 tip onto a p1000 tip and use this double tip for pipetting.</li>
	<li>Add 1 ml of SDS extraction solution to the cartridge filter unit so the total volume is about 2 ml and incubate for 10 min&#160;at room temperature while rotating in a hybridization oven. Place 3 crumpled lab wipes at the bottom of a 50 ml conical tube. Parafilm both ends of the cartridge filter unit closed and put the cartridge filter unit into the 50 ml tube. Close the lid and put the tube into the hybridization oven.</li>
	<li>After 10 min&#160;place the Sterivex filter on a foam floater and secure it in place with a 5 ml syringe on the luer-lock end. Float and incubate in a 95 &#176;C water-bath for 15 min.</li>
	<li>Let the cartridge filter unit cool and rotate at room temperature for 1 hour (as in step 2.6).</li>
	<li>Expel the SDS extraction solution/cell lysate out of the filter with a 60 ml syringe into the same ultracentrifugal filter unit as before (step 2.3). Add 1 ml of fresh SDS extraction solution to the cartridge filter unit and mix for 30 sec&#160;by hand, then expel into the ultracentrifugal filter unit using the 60 ml syringe. This is to rinse the cartridge filter unit to ensure all proteins have been removed.</li>
	<li>Perform centrifugation on the ultracentrifugal filter unit for 45 min&#160;at 3,270 x g or until the volume in the filter unit is less than 600 &#181;l.</li>
	<li>Discard flow-through and top up the ultracentrifugal filter unit with fresh SDS-extraction solution.</li>
	<li>Centrifuge the filter unit for another 45 min&#160;at 3,270 x g.</li>
	<li>Repeat steps 2.11 and 2.12 twice more. Ensure the final volume in the ultracentrifugal filter unit is at most 600 &#181;l at the end of the final spin.</li>
	<li>At this point, split the concentrate in two. One fraction will be used for DNA precipitation and the other for protein precipitation. 
	Note: The fractionation amount depends on the amount of biomass that was filtered and the intended uses of the products. In our case, we split the concentrate with 10% towards DNA precipitation and 90% towards protein precipitation.</li>
</ol>
3. Protein Precipitation
<ol>
	<li>Add 4 volumes methanol:acetone (50:50) to one volume of concentrate and vortex for 10 sec. Incubate overnight at -20 &#176;C.</li>
	<li>Spin down at 17,000 x g for 30 min. Decant the supernatant and let the pellet (may be invisible) dry in a speedvac for 1 hr (or until dry). 
	Note: Do not over dry the pellet as this may make it difficult to resuspend.</li>
	<li>Suspend the pellet in 25 &#181;l of SDS-extraction solution. Let sit for one hour then resuspend by pipetting up and down.</li>
	<li>Quantify protein using a protein assay kit and the manufacturers instructions.</li>
</ol>
4. DNA Precipitation
<ol>
	<li>Add SDS-extraction solution to the concentrate fraction to be used for DNA precipitation until the 500 &#181;l mark. This step is simply to increase the volume of the solution, making it easier to work with.</li>
	<li>Add 0.583 volumes of a protein precipitation reagent (such as the MPC protein precipitation reagent) and vortex for 10 sec. You should see a white precipitate form. 
	Note: We have also used phenol:chloroform DNA extraction methods, but it is more difficult due to the low volumes. We obtained better DNA yields using the MPC Protein Precipitation Reagent.</li>
	<li>Centrifuge at 17,000 x g and 4 &#176;C for 10 min.</li>
	<li>Transfer supernatant to another 1.5 ml microcentrifuge tube and add 0.95 volumes of isopropanol. Invert 30-40 times.</li>
	<li>Centrifuge for 10 min at 4 &#176;C at maximum speed.</li>
	<li>Carefully decant and discard the supernatant.</li>
	<li>Rinse twice with 750 &#181;l of 70% ethanol.</li>
	<li>Remove as much ethanol as possible by pipetting, then let air-dry. 
	Note: Do not over dry the DNA as this may make it difficult to resuspend.</li>
	<li>Resuspend in 25 &#181;l of low TE buffer,&#160;pH 8&#160;(10 mM Tris-HCl, 0.1 mM EDTA).</li>
	<li>Quantify the DNA using a dsDNA assay kit and the manufacturers instructions. Perform agarose gel (1%) electrophoresis on 3 &#181;l of the DNA to check its quality.</li>
</ol>
5. SDS-PAGE Gel of Proteins
<ol>
	<li>Prepare sample buffer (950 &#181;l Laemmli sample buffer and 50 &#181;l &#946;-mercaptoethanol).</li>
	<li>Add the corresponding volume for 15 &#181;g of protein, or a max of 20 &#181;l to equal volume of sample buffer and boil for 4 min. Samples can now be stored at -80 &#176;C until SDS-PAGE can be performed.</li>
	<li>Prepare 10% acrylamide resolving gel with a 5% acrylamide stacking gel.</li>
	<li>Load the gel with samples and 4 &#181;l of a protein ladder. Run the gel at constant 120 V until the 250 kDa ladder marker has just reached the resolving gel.</li>
	<li>Stain the gel using a coomassie based stain according to the manufacturers instructions.</li>
</ol>
6. In-gel Trypsin Digest and Peptide Extraction
Note: All steps from here until 6.10&#160;are performed in a biological safety cabinet to minimize contamination.
<ol>
	<li>Cut off excess gel under the 10 kDa ladder mark for each lane. Each lane will be analyzed on the MS/MS separately. Cut each lane into 1 mm x 1 mm squares to increase surface area and place all squares from the lane into a low-binding micro-centrifuge tube. Repeat this for all lanes. (1% acetic acid can be used to prevent the gel from drying out and becoming difficult to cut). 
	Note: Minimizing contamination is critical at this stage, so gel slicing is performed in a biological safety cabinet on the glass SDS-PAGE gel mold used to make the gel.</li>
	<li>De-stain
	<ol>
		<li>Dispense 50 &#181;l of 100 mM ammonium bicarbonate into each low-binding tube, close cap and incubate at 37 &#176;C for 10 min.</li>
		<li>Dispense 50 &#181;l of acetonitrile, close cap and incubate at 37 &#176;C for 5 min.</li>
		<li>Aspirate and discard 150 &#181;l.</li>
		<li>Repeat steps 6.2.1 and 6.2.2.</li>
		<li>Aspirate and discard 95 &#181;l.</li>
	</ol>
	</li>
	<li>Dehydration
	<ol>
		<li>Dispense 50 &#181;l acetonitrile, close cap and wait 5 min. Aspirate and discard 45 &#181;l and wait 10 min.</li>
	</ol>
	</li>
	<li>Reduction
	<ol>
		<li>Dispense 50 &#181;l DTT (10 mM, diluted in 100 mM ammonium bicarbonate), close cap and wait 30 min&#160;at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Alkylation
	<ol>
		<li>Dispense 50 &#181;l iodoacetamide (55 mM, diluted in 100 mM ammonium bicarbonate), close cap and wait 20 min&#160;at 37 &#176;C.</li>
		<li>Dispense 100 &#181;l of acetonitrile, close cap and incubate for 5 min&#160;at 37 &#176;C. Aspirate and discard 195 &#181;l.</li>
	</ol>
	</li>
	<li>Wash
	<ol>
		<li>Dispense 50 &#181;l ammonium bicarbonate, close cap and incubate for 10 min&#160;at 37 &#176;C.</li>
		<li>Dispense 50 &#181;l of acetonitrile, close cap and incubate at 37 &#176;C for 5 min. Aspirate and discard 120 &#181;l.</li>
	</ol>
	</li>
	<li>Dehydration
	<ol>
		<li>Dispense 50 &#181;l acetonitrile, close cap and wait 5 min. Aspirate and discard 45 &#181;l.</li>
		<li>Dispense 50 &#181;l of acetonitrile, wait 5 min.</li>
		<li>Aspirate and discard 75 &#181;l and wait 5 min.</li>
	</ol>
	</li>
	<li>Digestion
	<ol>
		<li>Dispense 25-30 &#181;l of 6 ng/&#181;l Trypsin (until all gel fragments are covered). Close cap and wait 30 min&#160;at room temperature.</li>
		<li>Incubate overnight (4.5 hr&#160;minimum) at 37 &#176;C.</li>
		<li>Let sit at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Extraction and Peptide Transfer
	<ol>
		<li>Dispense 30 &#181;l of extraction solution and cover with lid for 30 min&#160;on ice.</li>
		<li>Aspirate 30 &#181;l and dispense aspirated volume in a new labeled low-binding tube.</li>
		<li>Dispense 12 &#181;l of extraction solution and 12 &#181;l of acetonitrile into the original tube (with gel). Close cap and incubate for 30 min&#160;on ice.</li>
		<li>Aspirate 15 &#181;l and deposit into the new tube.</li>
		<li>Repeat steps (6.9.3 and 6.9.4).</li>
	</ol>
	</li>
	<li>Place new tubes in a SpeedVac until dry.</li>
	<li>Resuspend in 50 &#181;l of resuspension solution. Vortex on medium for 10 min&#160;to resuspend.</li>
	<li>Pipette peptide solution into either nano/LC vials or 96-well plates suitable for nano/LC MS/MS work.</li>
</ol>

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<li>Thomas, T., Gilbert, J., Meyer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metagenomics+-+a+guide+from+sampling+to+data+analysis%5BTitle%5D)+AND+%22Microbial+Informatics+and+Experimentation%22%5BJournal%5D)">Metagenomics - a guide from sampling to data analysis.</a> Microbial Informatics and Experimentation. 2 (1), 3(2012).</li>
<li>Huson, D. H., Auch, A. F., Qi, J., Schuster, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MEGAN+analysis+of+metagenomic+data%5BTitle%5D)+AND+%22Genome+research%22%5BJournal%5D)">MEGAN analysis of metagenomic data.</a> Genome research. 17 (3), 377-3786 (2007).</li>
<li>Huson, D. H., Mitra, S., Ruscheweyh, H. -J., Weber, N., Schuster, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrative+analysis+of+environmental+sequences+using+MEGAN4%5BTitle%5D)+AND+%22Genome+research%22%5BJournal%5D)">Integrative analysis of environmental sequences using MEGAN4.</a> Genome research. 21 (9), 1552-1560 (2011).</li>
<li>Ea Ottesen,, Marin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metatranscriptomic+analysis+of+autonomously+collected+and+preserved+marine+bacterioplankton%5BTitle%5D)+AND+%22The+ISME+journal%22%5BJournal%5D)">Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton.</a> The ISME journal. 5 (12), 1881-1895 (2011).</li>
<li>Feike, J., Jürgens, K., Hollibaugh, J. T., Krüger, S., Jost, G., Labrenz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+unbiased+metatranscriptomics+in+suboxic+waters+of+the+central+Baltic+Sea+using+a+new+in+situ+fixation+system%5BTitle%5D)+AND+%22The+ISME+journal%22%5BJournal%5D)">Measuring unbiased metatranscriptomics in suboxic waters of the central Baltic Sea using a new in situ fixation system.</a> The ISME journal. 6 (2), 461-470 (2012).</li>
</ol>

The authors have nothing to disclose.

Sample preservation is key to metaproteomic studies and previous work demonstrated that an RNA stabilization solution is a useful storage buffer for storing cells prior to protein extraction 28. Ideally, samples would be preserved in situ to negate shifts in protein expression during handling 33,34. In fact, in situ sampling and fixation technologies have been developed, which allow for the autonomous collection and preservation of samples by ship-deployed instruments. However, acc...

Microorganisms are ubiquitous and play essential roles in Earth&#8217;s biogeochemical cycles 1. Currently, there are numerous molecular approaches available for characterizing microbial community structure and function. Most common is the analysis of 16S rRNA gene sequences PCR-amplified from environmental DNA 2&#8211;4. A disadvantage of 16S rRNA gene analysis is that it only provides information on phylogenetic identity and community structure, with little information on metabolic function. In contrast, approaches such as metagenomics, metatranscriptomics and metaproteomics provide information on community structure and metabolism. Metagenomics, or the analysis of the gene content of an assemblage of organisms, provides information about the structure and functional potential of the community 5&#8211;8. Although powerful, this functional potential may not correspond to the metabolic activities of the organisms. An organism&#8217;s genotype is represented by its genes, each of which can be transcribed to RNA and further translated to protein, resulting in a phenotype. Thus, to aid in the understanding of microbial functional activity in an environment, post-genomic analysis should be performed 9. Metatranscriptomics, or the analysis of RNA transcripts is useful because it reveals which genes are transcribed in any given environment. However, mRNA levels do not always match their corresponding protein levels due to translational regulation, RNA half-life, and the fact that multiple protein copies can be generated for every mRNA 10.
For these reasons metaproteomics is now recognized as an important tool for environmental microbiology. Common metaproteomic analyses use a shotgun proteomic approach where the near full complement of proteins in a complex sample are purified and analyzed simultaneously, usually through enzymatic digestion into peptides and analysis on a mass spectrometer. Subsequent tandem mass spectrometry (MS/MS) &#8220;peptide fingerprinting&#8221; is used to determine the peptide sequence and potential protein of origin by protein database searching (for a review see 11). Proteomic work has come a long way in the past 25 years thanks to the increase in genomic data availability and the increase in the sensitivity and accuracy of mass spectrometers allowing for high-throughput protein identification and quantification 11,12. Since proteins are the final product of gene expression, metaproteomic data can help determine which organisms are active in any given environment and what proteins they are expressing. This is advantageous when trying to determine how a particular set of environmental variables will affect the phenotype of an organism or community. Early on, MS/MS-based metaproteomic studies in the ocean were used to identify specific proteins in targeted microbial lineages, with the first study focusing on the light driven proton pump proteorhodopsin in SAR11 marine bacteria 13. More recently, comparative metaproteomic analyses have elucidated differential protein expression patterns between complex communities. Examples include the identification of temporal shifts in metabolism in the coastal Northwest Atlantic Ocean 14 or the Antarctic Peninsula 5. Other studies have described variations in protein expression patterns across spatial scales, for instance, along a geographical transect from a low-nutrient ocean gyre to a highly productive coastal upwelling system 15. For further reviews of metaproteomics we recommend Schneider et al. (2010) 9 and Williams et al. (2014) 16. Targeted proteomics has also been employed in recent years to quantify expression of specific metabolic pathways in the environment 17,18.
There are three main phases in metaproteomic analysis. The first phase is sample preparation, which includes sample collection, cell lysis and concentration of protein. Sample collection in marine microbiology often entails the filtration of seawater through a pre-filter to remove larger eukaryotic cells, particles and particle-associated bacteria, followed by filtration for the capture of free living microbial cells, commonly with the use of a 0.22 &#181;m cartridge filter unit 19,20. These filters are incased in a plastic cylinder and a cell lysis and protein extraction protocol that can be performed within the filter unit would be a valuable tool. Once biomass is obtained, the cells must be lysed to allow for protein extraction. Several methods can be employed, including guanidine-HCl lysis 21 and sodium dodecyl sulfate (SDS)-based lysis methods. Although detergents like SDS are very efficient at disrupting membranes and solubilizing many protein types, concentrations as low as 0.1% can interfere with downstream protein digestion and MS analysis 22. Of major concern is the negative effects of SDS on trypsin digestion efficiency, resolving power of reverse phase liquid chromatography and ion suppression or accumulation inside the ion source 23.
The second phase is fractionation and analysis, where proteins are subjected to enzymatic digestion followed by LC MS/MS analysis, resulting in a m/z fragmentation pattern that can be used to ascertain the primary amino acid sequence of the initial tryptic peptide. Various digestion methods can be performed depending on the types of detergents used, as well as the downstream mass spectrometry workflow. In our protocol, 1-D PAGE electrophoresis followed by removal of SDS from the gel is utilized in order to remove any detergent contamination. The analysis of proteins that are difficult to solubilize, such as membrane proteins, requires the use of high concentrations of SDS or other detergents. This leads to compatibility issues with SDS-gel electrophoresis. If the objective of a study requires the solubilization of these hard to solubilize proteins, the tube-gel system can be used 22,24. The tube-gel method incorporates proteins within the gel matrix without the use of electrophoresis. Subsequently any detergents used for solubilization are removed before protein digestion.
The third phase is the bioinformatic analysis. In this phase the MS/MS peptide data are searched against a database of translated nucleotide sequences to determine which peptides and proteins are present in the sample. The identification of peptides is dependent on the database it is searched against. Marine metaproteomic data are commonly searched against databases comprised of reference genomes, metagenomic data such as the Global Ocean Sampling dataset 25, as well as single cell amplified genomes from uncultivated lineages 26,27. Protein identification can also be increased by the inclusion of metagenomic sequences from the same sample as the metaproteomic data was derived 5.
Here we provide a protocol for the generation of peptides suitable for MS/MS-based analysis from microbial biomass collected by filtration and stored in an RNA stabilization solution. The protocol described here allows for DNA and protein to be isolated from the same sample so that all steps leading up to the protein and DNA precipitations are identical. From a practical perspective, less filtration is required since only one filter is required for both protein and DNA extraction. We would also like to acknowledge that this protocol was created through the combination, adaptation and modification of two previously published protocols. The cell lysis steps are adapted from Saito et al. (2011) 28 and the in-gel trypsin digest component is adapted from Shevchenko et al. (2007) 29.

<table><tbody><tr><td>Sterivex -GP 0.22 &amp;mu;m filter unit</td><td>Millipore</td><td>SVGP01050</td><td>Sampling</td></tr><tr><td>RNAlater Stabilization Solution</td><td>Ambion</td><td>AM7021</td><td>Sampling</td></tr><tr><td>Tris&amp;nbsp;</td><td>Bio Basic</td><td>77-86-1 or TB0196-500G</td><td>Protein Extraction/ SDS PAGE gel</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632-1G</td><td>Protein Extraction</td></tr><tr><td>SDS</td><td>Bio Basic</td><td>15-21-3</td><td>Protein Extraction/ SDS PAGE gel</td></tr><tr><td>EDTA</td><td>Bio Basic</td><td>6381-92-6</td><td>Protein Extraction</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td>Protein Extraction</td></tr><tr><td>10K Amicon Filter</td><td>Millipore</td><td>UFC801024</td><td>Protein Extraction</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179337-4L</td><td>Protein Precipitation</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>67-64-1</td><td>Protein Precipitation</td></tr><tr><td>MPC Protein Precipitation reagent</td><td>Epicenter</td><td>mmP03750</td><td>DNA Precipitation</td></tr><tr><td>2-Propanol</td><td>Fisher Scientific</td><td>67-63-0</td><td>DNA Precipitation</td></tr><tr><td>Qubit dsDNA BR Assay kit</td><td>Life Technologies</td><td>Q32850</td><td>DNA Quantification</td></tr><tr><td>Qubit Protein Assay kit</td><td>Life Technologies</td><td>Q33211</td><td>Protein Quantification</td></tr><tr><td>Sucrose</td><td>Bio Basic</td><td>57-50-1</td><td>SDS PAGE gel</td></tr><tr><td>TEMED</td><td>Bio Rad</td><td>161-0800</td><td>SDS PAGE gel</td></tr><tr><td>APS</td><td>Bio Rad</td><td>161-0700</td><td>SDS PAGE gel</td></tr><tr><td>30% Acrylamide</td><td>Bio Rad</td><td>161-0158</td><td>SDS PAGE gel</td></tr><tr><td>SimplyBlue SafeStain</td><td>Invitrogen</td><td>LC6060</td><td>SDS PAGE gel</td></tr><tr><td>Glycine</td><td>Bio Rad</td><td>161-0718</td><td>SDS PAGE gel</td></tr><tr><td>B-mercaptoethanol</td><td>Bio Basic</td><td>60-24-2</td><td>SDS PAGE gel</td></tr><tr><td>Laemmli Sample Buffer</td><td>Bio Rad</td><td>161-0737</td><td>SDS PAGE gel</td></tr><tr><td>Precision Plus Protein Kaleidoscope Ladder</td><td>Bio Rad</td><td>161-0375EDU</td><td>SDS PAGE gel</td></tr><tr><td>Acetonitrile</td><td>VWR</td><td>CABDH6044-4</td><td>In-gel Trypsin digest</td></tr><tr><td>NH4HCO3</td><td>Bio Basic</td><td>1066-33-7</td><td>In-gel Trypsin digest</td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>D0632-1G</td><td>In-gel Trypsin digest</td></tr><tr><td>Formic Acid</td><td>Sigma-Aldrich</td><td>F0507-500ML</td><td>In-gel Trypsin digest</td></tr><tr><td>HPLC grade H2O</td><td>Sigma-Aldrich</td><td>270733-4L</td><td>In-gel Trypsin digest</td></tr><tr><td>Iodoacetamide</td><td>Bio Basic</td><td>144-48-9</td><td>In-gel Trypsin digest</td></tr><tr><td>Trypsin</td><td>Promega</td><td>V5111</td><td>In-gel Trypsin digest</td></tr><tr><td>Protein LoBind Tube 1.5 ml</td><td>Eppendorf</td><td>22431081</td><td>In-gel Trypsin digest</td></tr><tr><td>2 ml ROBO vial 9 mm</td><td>Candian Life Science</td><td>VT009/C395SB</td><td>In-gel Trypsin digest</td></tr><tr><td>PP BM insert, No spring</td><td>Candian Life Science</td><td>4025P-631</td><td>In-gel Trypsin digest</td></tr></tbody></table>

an aquatic microbial metaproteomics workflow: from cells to tryptic peptides suitable for tandem mass spectrometry-based analysis

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

As a demonstration, we performed the protocol on two seawater samples collected from the surface and the chlorophyll maximum of the coastal ocean in Northern Canada. While at sea, 6-7 L of seawater was passed through a 3 µm GF/D prefilter, then microbial cells were collected onto a 0.22 µm cartridge filter unit following the protocol of Walsh et al. 20. Cells were immediately stored in an RNA stabilization solution until further processing. Upon returning to the lab, we performed the protocol as it is...

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

The goal of this procedure is the extraction of protein and DNA from microbial biomass from seawater, followed by the generation of triptic peptides for tandem mass spectrometry based proteomic analysis. This is accomplished by first lysing cells previously accumulated on a cartridge filter. The second step is to concentrate the cell lysate using ultra centrifugal filter units.Next, the lysate is separated into two fractions, one for protein precipitation and one for DNA precipitation. The final step is to perform ingel trypsin digestion of proteins and extract peptide suitable for liquid chromatography, tandem mass spectrometry analysis. Ultimately, subsequent bioinformatic analysis can be used to identify expressed proteins in the sample while the isolated DNA can be used for metagenomic and or 16 S-R-R-N-A gene analysis.The main advantage of this technique over existing methods is that both protein and DNA can be isolated from the same sample simultaneously. To begin expel the RNA stabilization solution from the cartridge filter unit and into a two milliliter micro centrifuge tube, using a 60 milliliter syringe attached at the lure lock end of the filter centrifuge, the two milliliter micro centrifuge tube for 10 minutes at 17, 000 times, gravity to pellet any cellular debris following centrifugation. Transfer the supra natin to a 10 K ultra centrifugal filter unit to capture any proteins originating from cells that have lyed from the sample.Do not discard the pellet. Perform centrifugation of the ultra centrifugal filter unit at 3, 270 times gravity for 30 minutes or until the volume has reduced to about 600 microliters. Meanwhile, melt the tip of a P 10 tip and block the non lure lock side of the cartridge filter unit with the open end of the tip.This will ensure that no extraction buffer and biomass escape during future steps To suspend the pellet in one milliliter of SDS extraction solution and pipette it into the original cartridge filter unit. The easiest way to pipette into the cartridge filter unit is to stack a P 200 tip onto a P 1000 tip and use this double tip for pipetting. Add an additional one milliliter of SDS extraction solution to the cartridge filter unit so that the total volume is about two milliliters.Para film. The lure lock end of the cartridge filter unit closed and put the cartridge filter unit into the 50 milliliter tube. Next place three crumpled lab wipes at the bottom of a 50 milliliter conical tube.Close the lid and put the tube into the hybridization oven to incubate for 10 minutes at room temperature while rotating After 10 minutes, place the filter on a foam floater and secure it in place with a five milliliter syringe on the lure lock end load an incubate in a 95 degree Celsius water bath for 15 minutes. Then let the cartridge filter unit cool and rotate at room temperature for one hour.Once.Cool. Expel the SDS extraction solution cell lysate from the filter with a 60 milliliter syringe into the same ultra centrifugal filter unit.As before, add one milliliter of fresh SDS extraction solution to the cartridge filter unit and mix for 30 seconds by hand. Then expel the contents of the filter into the ultra centrifugal filter unit using the 60 milliliter syringe to rinse and ensure all proteins have been removed and collected. Perform centrifugation on the ultra centrifugal filter unit for 45 minutes at 3, 270 times gravity, or until the volume in the filter unit is less than 600 microliters.Discard the flow through and top up the ultra centrifugal filter unit with fresh SDS extraction solution. Centrifuge the filter unit for another 45 minutes at 3, 270 times gravity before repeating this step twice more. Ensure the final volume in the ultra centrifugal filter unit is at most 600 microliters at the end of the final spin.At this point, measure the volume of the concentrate and split the concentrate in two. One fraction will be used for DNA precipitation and the other for protein precipitation. For protein precipitation, add four volumes of methanol and acetone in a 50 to 50 ratio to one volume of concentrate and vortex for 10 seconds, incubate overnight at minus 20 degrees Celsius.The next day, spin down the mixture at 17, 000 times. Gravity for 30 minutes after decanting the super named, let the pellet dry in a speed vac for one hour. Do not over dry the pellet as this may make it difficult to resend.Suspend the pellet in 25 microliters of SDS extraction solution. Let it sit for one hour and then resuspend by pipetting up and down. Perform SDS page on the proteins as detailed in the text protocol working in a biological safety cabinet to minimize contamination, cut off the excess gel under the 10 kilodalton ladder mark for each lane.Each lane will be analyzed on the tandem mass spectrometer. Separately, cut each lane into one millimeter by one millimeter squares to increase surface area and place all squares from the lane into a low binding micro centrifuge tube. Repeating for all lanes 1%Acetic acid can be used to prevent the gel from drying out and becoming difficult to cut.Proceed with in gel trypsin digest and peptide extraction as described in the text protocol or DNA precipitation at SDS extraction solution to the concentrate fraction to be used for DNA precipitation until the 500 microliter mark. This step is simply to increase the volume of the solution, making it easier to work with. Next at 0.583 volumes of a protein precipitation reagent such as the MPC protein, precipitation, reagent, and vortex.For 10 seconds, a white precipitation form centrifuge at 17, 000 times gravity and four degrees Celsius for 10 minutes. Following centrifugation, transfer the supinate into another 1.5 milliliter micro centrifuge tube and add 0.95 volumes of isopropanol invert 30 to 40 times before centrifuging for 10 minutes at four degrees Celsius at maximum speed, carefully decant and discard the supernatant. Then rinse the pellet twice with 750 microliters of 70%ethanol.Remove as much ethanol as possible by pipetting, and then let the pellet air dry. Do not over dry the DNA as. This may make it difficult to resend once dry Resus, suspend the pellet in 25 microliters of low T buffer P eight.Quantify the DNA using a double stranded DNA assay kit and the manufacturer's instructions. Also perform agarose gel electrophoresis on three microliters of the DNA to check its quality peptides were subjected to tandems spectrometry analysis from the peptides. Over 23, 000 spectral were generated per sample from which peptides and proteins were identified.Taxonomic and functional composition of the meta proteomes was analyzed using a combination of blast P and the metagenome analyzer. Software package proteins assigned to alpha proteobacteria were the most highly represented in the dataset. The vast majority of which were assigned to the SAR R 11 clade.The Roter clade of Alpha Proteobacteria was also highly represented and identified. Most often in surface waters. Proteins assigned to bacter disease were evenly distributed between the surface and chlorophyll maximum, but flail bacteria proteins were identified to a greater degree at the chlorophyll.Maximum.Gamma proteobacteria proteins were evenly distributed throughout the water column while beta protio bacterial proteins were found predominantly in the surface Once mastered. This technique can be done in three days if performed properly.

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JoVE Journal

Environment

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.

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

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.

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

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

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.

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

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of disease.

A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Learn an aquatic microbial metaproteomics workflow for extracting proteins and DNA from seawater samples. This method generates tryptic peptides suitable for tandem mass spectrometry analysis.

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis (Video) | JoVE Journal

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

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