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

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.