The overall goal of the following experiment is to separate different membrane sub compartments and study their differential protein composition. This is achieved by stable isotope labeling of cells under different conditions to allow for differentiation of the treatments. In mass spectrometric analysis as a second step cell material from the two treatments is mixed, which ensures joint processing and reduces sample to sample variation.
Next plasma membrane sub compartments are purified and separated into sterile, rich and sterile depleted sub compartments by sucrose. Gradient centrifugation results are obtained that show differential protein abundances in the sub compartments under different treatments based on data from quantitative mass spectrometry. The main advantage of this protocol over existing methods, such as Western blotting, is that we can quantitatively record the sub compartment distribution of thousands of proteins and thereby discover new candidates for signaling processes.
Visual demonstration of this method is critical as the me purification of a two phase system is difficult to learn. The reason for that is that it involves a lot of steps and the precise controlling and thorough handling is very important. This method can help answer key questions in signaling field by discovering new protein candidates as regulators of membrane processes Begin by growing labeled and unlabeled arabidopsis ana cell suspension cultures as described in the text protocol.
Homogenized cells from about one liter of seven day old cell suspension cultures in liquid nitrogen mix the same amounts of labeled and unlabeled frozen cell cultures based on fresh weight in a beaker and add two volumes of buffer H, immediately place the beaker on a shaker and shake until the material is melted and without ice. Crystals filter the homogenate through one layer of mirror cloth into 250 milliliter centrifuge tubes after balancing the samples with buffer H centrifuge at 10, 000 Gs for eight minutes. Following centrifugation, load the snat into pre-cool ultra centrifuge tubes with a volume of about 32 milliliters.
After balancing the samples with buffer h pellet the microsomes by centrifuging at 100, 000 Gs and four degrees Celsius for 30 minutes. After centrifugation remove the supernatant, the supernatant contains soluble proteins. A small amount of this fraction can be saved for further protein precipitation, and subsequent analysis of soluble proteins re suspend the membrane pellet of each sample in the respective amount of buffer R as described in the text protocol load onto the top of the U one two phase system.
If the six gram two-phase system is used, load exactly two grams of resuspended membrane pellet load the same volume of pure buffer R as used for the sample resus suspension onto U2.Slowly mix U one and U2 tubes by inverting them 30 times at approximately one inversion per second centrifuge the samples at 1500 Gs and four degrees Celsius for 10 minutes. After centrifugation, remove the upper phase from U2.Transfer the upper phase from U one onto U2 before repeating centrifugation using the same parameters. Next, move the upper phase from U2 into the ultra ultracentrifuge tubes dilute with five volumes of buffer R and mix thoroughly centrifuge the samples at 200, 000 Gs and four degrees Celsius for one hour.
Finally, reuse. Suspend the membrane pellet in a small amount of T and E buffer for isolation of detergent resistant membranes. After checking the concentration of the plasma membrane fraction by Bradford assay, add Triton X 100 to the plasma membrane fraction at a detergent to protein, weight to weight ratio between 13 and 15, and a final detergent concentration between 0.5 and 1%Shake the samples at around 100 RPM and four degrees Celsius for 30 minutes.
Then add three volumes of 2.4 molar sucrose to one volume of the treated plasma membrane to obtain a 1.8 molar final concentration of sucrose. Prepare the sucrose gradient in ultracentrifuge tubes by loading the respective sucrose solutions on top of the 1.8 molar fraction Centrifuge the samples at 250, 000 GS and four degrees Celsius for 18 hours. Carefully remove the ultracentrifuge tubes from the rotor to avoid disruption of the low density band, which may be visible as a milky ring at the interface of 0.15 molar and 1.4 molar sucrose.
Sometimes nothing can be seen, but the detergent resistant protein fraction is still there. Remove a fraction of 0.75 milliliter volume from the top of the gradient, fractions two and three, which cover the volumes of around 1.5 milliliters above the interphase ring. And 0.5 milliliters below the interphase ring.
Represent the detergent resistant membrane fraction. Collect these fractions into 15 milliliter Falcon tubes. Fractions nine and 10 contain the detergent soluble membrane fraction and may also be collected for comparison.
To begin extraction of proteins from the detergent resistant fraction, add four volumes of methanol to the collected fractions and v vortex thoroughly. Then add one volume of chloroform before vortexing thoroughly again. Finally, add three volumes of water and vortex thoroughly centrifuge the samples at 2000 Gs for five.
Using a benchtop centrifuge, remove the aqueous phase above the protein layer in the interphase. Next, add three volumes of methanol and vortex. Thoroughly remove the S supernatant before drawing the pellet To prepare for insolution digestion.
Begin insolution digestion by dissolving the sample in a small volume of six molar urea two molar thyroid pH eight abbreviated UTU. Use as low a volume as possible that is compatible with the sample. After spinning the samples and performing the digestion as described in the text protocol, dilute the sample with four volumes of 10 millimolar tris HCL PH eight.
Then add one microliter of trypsin per 50 micrograms of sample protein incubate overnight at room temperature with constant shaking at 700 RPM centrifuge the samples at 4, 000 GS for 10 minutes. Finally, acidify the samples to 0.2%TFA final concentration to reach pH two by adding approximately one 10th volume of 2%tri fluoro acetic acid. To manufacture C 18 stage tips, place an mour disc C 18 on a flat clean surface such as a disposable plastic Petri dish.
Punch out a small disc using a blunt tip hypodermic needle with a diameter of 1.5 millimeters. The disc sticks in the needle and can be transferred into a pipette tip. Push the disc out of the needle and fix it in the tapering of a pipette tip using a piece of fused silica or tubing fitting in the inside of the needle condition a C 18 stage tip by placing 50 microliters of solution B onto the prepared stage tip.
Spin the tip in the centrifuge at 2000 RPM for two minutes In a benchtop centrifuge, equilibrate the stage tip using 100 microliters of solution a. Spin the tip at 5, 000 GS in a benchtop centrifuge. Repeat the equilibration and spin.
Load the sample onto the disc by carefully pipetting the sample into the pipette tip with the disc inside. Spin the tips in the centrifuge until the whole volume of sample passes through the C 18 disc. Wash the stage tips two times with 100 microliters of solution A.After spinning the tips in the centrifuge, collect the EIT in a fresh 1.5 milliliter reaction tube at 40 microliters of solution B and centrifuge.
Again, concentrate the sample in the speed vac under ideal conditions. Stop the dehydration process when there is just about one or two microliters of liquid left. As a final step, add a final volume of nine microliters of Resus suspension solution to the sample and transfer it to the microtiter plate for mass spec analysis.
Enrichment of plasma membrane proteins can be shown by proteomic analysis of small eloqua of plasma membrane, fractions and proteins in the lower phase of the two-phase system. The lower phase contains the inner membranes of the cell. For example, typically known plasma membrane proteins such as receptor kinase proteins shown high abundance in the plasma membrane fraction and low abundance in the lower phase fraction containing the internal membranes.
In turn known proteins of the endoplasmic reticulum show high abundance in the inner membrane fraction and were not observed in the plasma membrane fraction. The subsequent enrichment of detergent resistant membrane fractions will, in ideal cases, result in formation of a milky ring of low density membrane vesicles. At approximately one fifth of the tube height, the highest abundance of remn protein was found in the detergent resistant membrane or DRM fractions as expected.
Conversely, proteins that are not present in the membrane, micro domains such as an A b, C transporter do not show an increased abundance in the DRM fraction. Also, typical contaminants such as a mitochondrial protein from the A TPAs F zero complex and ribosomal protein from the 60 s ribosome do not show an enriched abundance in the DRM fraction. Although minimal amounts can still be identified after mass spectrometric analysis, protein abundance ratios of protein phosphorylation status in plasma membrane, fractions of labeled and unlabeled cell cultures can be quantitatively distinguished.
Typical results from the MS quant quantitation window should show an ion intensity ratio of one-to-one for most of the identified peptides. Those peptides with ion intensity ratio significantly deviating from one-to-one are considered as differentially regulated between the labeled and unlabeled cell culture, and are candidates for matching to proteins affected by the treatment applied. A good quality control of successful metabolic labeling is the coalition of both labeled and unlabeled peptide versions as a single peak in the nano HPLC separation as shown here.
Once mastered, this technique can be done in three days from the start of the extraction till the sample loading onto mass spectrometer. Following this procedure, other methods like Western Belt can be performed in order to answer additional questions about supplication of specific target proteins. So after watching this video, we should have a good understanding of how to mix stable isotope labeled plant cell cultures, how to carry out a plasma membrane purification over two phase system, and how to separate sterile rich from sterile depleted membrane sub compartments.
