This protocol describes a method that enables site-specific identification of oxidized cystine residues of proteins. The method allows quantitative and site-specific data of oxidized cystine residues to be generated from various sample types. To begin the assisted capture procedure, wash the precipitated protein pellets twice with acetone by centrifuging at 20, 500 G, in four degrees Celsius for 10 minutes.
Then decant the acetone and remove the remaining acetone with a micro pipette. Using a glass serological pipette add three milliliters of fresh ice cold acetone to the tube and mix well by inverting the tube several times. After the second wash, allow the pellets to air dry for one to two minutes without letting them over dry.
Add one milliliter of buffer B to the dried protein pellet. To solubilize the protein, sonicate the pellet repeatedly in a bath sonicator with an output of 250 watts for 15 to 30 seconds at a time, along with brief vortexing. Perform the Bicinchoninic acid or BCA assay to measure the protein concentration.
To standardize the protein concentrations, transfer 500 micrograms of protein sample to a 0.5 milliliter, 10 kilodaltons centrifugal filter, and volume up the sample to 500 microliters with resuspension buffer. Centrifuge the protein buffer mixture at 14, 000 G in room temperature, until the volume in the centrifugal filter is less than 100 microliters. Collect the sample by inverting the filter in a collection tube.
Centrifuge the sample at 1000 G for two minutes and add buffer C to get a final volume of 500 microliters. To reduce the protein thiols, add 20 microliters of 500 millimolar dithiothreitol or DTT, to the sample to get a final concentration of 20 millimolar, and incubate the samples for 30 minutes at 37 degrees Celsius with shaking at 850 RPM. After reduction, centrifuge to the samples into 0.5 milliliter 10 kilodalton centrifugal filters at 14, 000 G at room temperature, until the volume in the centrifugal filter is less than 100 microliters.
Add buffer D to make up the volume to 500 microliters and repeat centrifugation. After the fourth centrifugation, collect the samples by inverting the filter in a collection tube to centrifuge at 1000 G for two minutes. Then measure the protein concentration using the BCA assay.
Next, place the spin columns on a vacuum manifold, and using a micro pipette transfer 500 microliters of the freshly prepared thiol affinity resin slurry to each column. Apply vacuum for removal of water. Repeat the procedure once to obtain 30 milligrams of resin per column.
Wash the resin five times with 500 microliters of ultrapure water, by applying a vacuum to remove the water and then five times with 500 microliters of buffer E.In a new tube, add buffer C to 150 micrograms of the reduced protein sample until the final volume is 120 microliters. Transfer the protein solution to a plugged spin column containing the resin. Place the cap on the column and incubate for two hours at room temperature with shaking at 850 rpm.
Wash the resin as described in the manuscript. Replace the plug. To perform on-resin tryptic digestion, add 120 microliters of freshly prepared trypsin solution to the samples, and incubate the column overnight at 37 degrees Celsius, with shaking at 850 RPM.
On the next day, wash the resin multiple times as described in the manuscript and replace the plug. Using a micro pipette, add 40 microliters of 100 millimolar Triethylammonium bicarbonate buffer to the washed resin. Then add 70 microliters of the dissolved tandem mass tag or TMT labeling reagent, and incubate for one hour at room temperature with shaking at 850 RPM.
Store any remaining TMT reagent at minus 80 degrees Celsius. Wash the resin and replace the plug. Elute the TMT labeled peptides by adding 100 microliters of 100 millimolar ammonium bicarbonate buffer at pH 8.0, containing 20 millimolar DTT, to the column, and incubating the column on a thermal mixer at room temperature for 30 minutes at 850 RPM.
After incubation, perform elution by placing the column on a vacuum manifold, applying the vacuum and eluting the samples into a five milliliter micro centrifuge tube. Repeat the step once, then add 100 microliters of 80%acetonitrile with 0.1%trifluoroacetic acid to the column. After incubating the column for 10 minutes at room temperature, elute the sample in the same five milliliter centrifuge tube.
Place the eluted samples in a vacuum concentrator until dry. Then store the dry peptides at minus 80 degrees Celsius until further use. The qualitative analysis of peptide samples from RAW 264.7 cells was carried out with SDS-PAGE, wherein samples were compared by determining different levels of oxidation due to treatments.
The separated proteins were subsequently probed by western blot to further investigate the oxidation levels of individual proteins. Reported ion intensities of cystine containing peptides were analyzed by liquid chromatography tandem mass spectrometry. The thiol oxidation stoichiometry of iTRAQ, labeled enriched and oxidized peptides at individual cystine site levels were quantified.
Carefully remove acetone without disturbing the protein pellet. Ensure that the protein pellet is completely solubilized and quantitated precisely, and that resin is accurately allocated to spin columns. Be sure to resuspend resin during the wash steps.
Further evaluation of enriched proteins and peptides may be performed by SDS-PAGE and staining, western blotting and mass spectrometry.
