The overall goal of this experiment is to observe the effects of pseudomona siro PV tomato on redox regulated proteins in tomato plants. First inoculate tomato plants with pseudomona siro PV PV tomato to induce pathogen infection and protein response. Proceed to stain with DIAM benzine to observe changes in reactive oxygen species.
Then perform cyst TMT labeling in order to examine redox regulated cysteines in proteins. Results obtained can measure changes between treated and control samples. Hi, I'm Chen from department biology and proteomics facility here at the University of Florida.
The advantage of our CTMT tagging over existing method such as isotope coated fin tag, also known ASCA, is the ability to multiplex while analyzing redox responsive proteins on large scale. While this demonstration will provide insights into tomato redox regulated proteins in response to pathogen infection, the experiment can be applied to other systems such as joof, mice cu, and other plants and control of stressed conditions. We first had the idea for this method after we observed marked variation between ICAD experiments because only two tags are available.
VIR demonstration of this method is critical as the labeling and enrichment steps are difficult to learn. Experimental variations between labeled samples must be minimized and reducing regions removed to ensure efficient labeling, Let's get started. Grow two transplanted tomato seedlings of similar size in four inch diameter pots for the inoculum culture.
A lawn of pseudomona singe on A KBM plate. Scrape the bacteria into 20 milliliters of water and prepare a bacteria inoculum of around 10 to the six colony forming units per milliliter. In one liter of inoculation buffer dip the four week old plants into the bacterial inoculation solution for 30 seconds.
Then cover immediately with clear plastic bags. After the preferred inoculation time, harvest the fully expanded leaf place epidermal side up in one milligram per milliliter, DAB stain and vacuum infiltrate for 15 minutes. Then incubate leaves in the dark at room temperature overnight.
Now boil the stained leaves in 95%ethanol for 10 minutes and store in 75%ethanol using a of mortar grind. The harvested leaves to a fine powder in liquid nitrogen. Then perform phenol extractions as described in the accompanying text.
Next, precipitate phenol extracted proteins with five volumes of cold 0.1 molar ammonium acetate in 100%methanol. Harvest the proteins by centrifugation, perform pellet washes. Then re suspend samples in 1.5 milliliters of cold, 70%ethanol and transfer to a two milliliter micro centrifuge tube.
After centrifugation, discard the ethanol supinate and dry the pellet briefly in a speed vac dissolve samples in a denaturing protein extraction buffer after centrifuging to form a pellet, collect the supinate and measure the supernatant and measure the protein concentration according to the manufacturer's manual. For each of the six mass tags, start with 100 micrograms of protein sample at equal volume of freshly prepared, 200 millimolar IO acetamide alkylation buffer to block the free thiol groups incubate at 37 degrees Celsius for an hour. Then to precipitate the proteins, add one milliliter of cold, 80%acetone and place a minus 20 degrees Celsius overnight.
Pellet the protein by centrifugation and perform three washes with 80%acetone. After air drying samples on ice, reus suspend the pellet in 50 microliters of lysis buffer. Then to reduce the diss sulfide bonds, add 0.5 microliters of 100 millimolar TCEP and incubate for one hour of room temperature.
Solubilize the tags by adding 20 microliters of aceto nitrile to the reagent tubes. Vortex and spin down Next to remove extra TCEP load 50 microliters of sample onto a MicroCon three KD column. Add 50 microliters of lysis, buffer and centrifuge for 15 minutes at 10, 000 times G and four degrees Celsius.
To collect the sample, remove the column invert into a clean tube and centrifuge. Check the pH of the sample if needed. Adjuster between pH seven and eight with one molar hydrochloric.
Add five micro release of the cyst TMT reagent and incubate the reaction for two hours of room temperature, combine samples and add an equal volume of laly sample buffer boil for five minutes and separate on a precast 12%poly acrylamide gel after rinsing the gel three times with filtered water stain with kumasi blue for an hour and detain overnight in water based on protein band concentrations. Divide 12 fractions per gel lane. Chop each fraction into one millimeter pieces and place into a 1.5 milliliter tube.
Completely detain the gel pieces with 0.1 molar ammonium bicarbonate in 50%aceto nitrile. Now cover the pieces with 100%aceto nitrile for 15 minutes. Remove the liquid and dry the pieces using a speed vac.
Next, add an equal volume of tripsin solution and set on ice to rehydrate. Then incubate a 37 degrees Celsius for 12 to 16 hours for triptych digestion. Next, remove the liquid protein extract from the incubated samples.
Stop the reaction and remove any remaining protein digest by covering the gel pieces with 5%formic acid, 50%aceto nitrile. Then shake the gel pieces for 20 minutes of room temperature. Transfer the supinate to the extracted protein sample tubes after two more extractions.
Dry the samples under a speed vac in 0.5 milliliter tubes. Prepare 50%slurry of anti TMT resin in TBS at a concentration proportional to the intensity of the respective protein band. Wash the resin three times with one column volume of one times TBS proceed to add 200 microliters of TBS to each sample.
Then transfer to anti TMT resin and agitate at room temperature for two hours, followed by rocking overnight of four degrees Celsius. Load each sample on a column. Perform column washes of 200 microliters each.
Then elute each sample three times with 200 microliters of 50%elution buffer and dry under vacuum. Reese, suspend the samples in 12 microliters of 3%aceto nitrile with 0.1%formic acid and inject five microliters directly onto an HPLC column. Elute peptides using a gradient of increasing aceto nitrile as detailed in the accompanying text.
Use mass spectrometry to detect peptides in a top two by three workflow. Start with a single stage ms. Then acquire three Ms.Ms spectra with higher energy C trapped dissociation fragmentation followed by three.
Ms.MS with collision induced dissociation for protein identification. Analyze the mass spec data using a branched workflow. Implement a reporter iron quantizer to quantify the ratio.
Also process the MSM MS spectrum through the spectrum selector spectrum normalizer and spectrum grouper nodes query data against the seaquest search engine. This image shows examples of a controlled tomato plant leaf on the left and a pseudomonas inoculated leaf on the right after the leaves are stained using DAB. The DES staining process allows for the histochemical stain to show signs of reactive oxygen species in the leaf tissue.
As expected the control shows no significant staining while the leaves treated with pseudomonas stains positive for hydrogen peroxide production. This example shows proteome discover data output of a differentially redox regulated protein peak area for each sample allows for absolute quantification. Peak area between control and inoculated samples indicates relative amounts.
Here, redox regulation of a protein between a control and inoculated sample is similar. Peaks of similar intensity suggests the presence of diss sulfide bonds not regulated by a change in treatment. Once mastered, this technique can be performed properly in one week.
Remember to keep samples cold and keep labels organized. Also use precautions when using phenol such as nag trial gloves and working in the Hood following this procedure. Other measure like specific mass spectrometry analysis of post translational modifications can be performed in order to determine other system modifications like onic acid and gluten simulation.
After watching this video, you should have a good understanding of how to perform DIB standing CTMD enabling and redox 16 qualification.
