The overall goal of this procedure is to use shotgun proteomics to characterize and quantify endogenous glutamine and asparagine deamidation products in complex proteomes. This method can help answer key questions in biochemistry, such as the influence of a spontaneous and antimatic processors on the isometric product gluteo of glutamine and asparagime deamination. The main advantage of this technique is that glutamine deamination isomers are separated on a long length ionic change capillary column, maximizing the ability to separate peptides by the isomatic points.
To begin column construction, suspend 50 milligrams of weak anion exchange packing material in 3.5 milliliters of 90%isopropanol, in water. Secure female to female fitting, a microferal, and a female nut at one end of a 50 centimeter length of peak tubing with an inner diameter of 200 micrometers. Fit a 1/16th inch screen with one micrometer pores at the end of the tubing inside the microferal.
Using pressure pump set to 4, 500 psi, pack the anion exchange into the capillary until the material is visible at the top. Attach another female-to-female fitting, microferal, and female nut to the top of the packed capillary at the opposite end to finish assembling the column. Wash 50 to 100 milligrams of tissue with phosphate buffered saline for five minutes.
Repeat this wash twice, and then place the washed tissue in tubes with safety caps. Add to each tube one weight equivalent of stainless steel beads, and 2.5 equivalents by volume of an aqueous 100 millimolar solution of ammonium acetate with 1%sodium deoxycholate. Homogenize the tissue at maximum intensity for five minutes at four degrees Celsius.
Then, centrifuge the homogenate for ten minutes at 10, 000 x g and four degrees Celsius. Transfer the supernatant to a 1.5 milliliter tube. Continue homogenizing and centrifuging the tissue pellet, combining the supernatants each time until no pellet is observed.
Quantify the protein concentration in the combined supernatants with a bicinchoninic acid assay. Next, add to the homogenate a one molar solution of dithiothreitol in 100 millimolar ammonium acetate to attain a ten millimolar dithiothreitol concentration in the sample. Incubate the homogenate in a water bath at 60 degrees Celsius for thirty minutes.
Add to the homogenate a one molar solution of iodoacetamide in 100 millimolar ammonium acetate to attain a 20 millimolar iodoacetamide concentration in the sample. Incubate the homogenate at room temperature in the absence of light for 45 minutes. Then dilute the homogenate with two volume equivalents of a ten millimolar solution of dithiothreitol in 100 millimolar ammonium acetate.
Incubate the homogenate at 37 degrees Celsius for 30 minutes. Add to the homogenate a sufficient volume of sequencing grade modified trypsin in 100 millimolar ammonium acetate to achieve a 1 to 50 protein enzyme ratio by weight in the sample. Incubate the sample at 30 degrees Celsius overnight to digest the homogenate.
Quench the digestion with 0.5%of the sample weight of formic acid, precipitating the sodium deoxycholate salts. Gently vortex the samples containing SDC salts and then centrifuge the samples for ten minutes at 12, 000 x g and four degrees Celsius. Decant the supernatant into a new tube, being careful not to disturb the pellet of SDC salts.
Redissolve the salts in a 0.5%by volume aqueous solution of ammonium hydroxide, vortexing vigorously. Condition a one gram C-18 sorbent cartridge with 5 milliliters of acetonitrile, followed by five milliliters of 0.1%trifluoroacetic acid in water. Then, load the digested sample onto the cartridge.
Wash the samples three to five times with five milliliter portions of 0.1%TFA. Then dilute the peptides with five milliliters of an aqueous solution of 75%acetonitrile, and 0.1%formic acid. Dry the fluid in a vacuum concentrator.
Add 200 microliters of 75%acetonitrile, with 0.1%formic acid to the residue. Vortex the mixture for at least ten minutes, and then sonicate the mixture for at least 30 minutes to reconstitute the dry peptides. Dilute the sample as needed, so that the injected sample contains one to three micrograms of protein.
Connect the LAX capillary column to an ultra high pressure liquid chromatography instrument. Prepare 0.1%formic acid solutions in water and acetonitrile. Set the flow rate to 0.4 microliters per minute, and set up a 1, 200 minute gradient run.
Configure the mass spectrometer scan events to alternate data acquisition, between full Fourier transform mass spectrometry and Fourier transform tandem mass spectrometry. Perform LERLIC MS/MS to separate and characterize the peptides. Use the resulting data and proteomic software to perform a protein database search.
Export the database search results to spreadsheet software. Identify and extract the list of deamidated peptides and the corresponding non-deamidated peptides. Extract the ion chromatagrams of each of these peptides with five parts per million of mass tolerance.
Inspect the extracted ion chromatagrams for separated double peak illusion of isomeric products. Using LERLIC MS/MS, deamidated isoforms of glutamine and asparagine were separated and identified from a protein sample at two different retention times. Enzymatically modified intermediate glutamine residues of transamidation showing an inverted gamma alpha glutamil ratio were identified.
The succinimide intermediate in asparagine residues was also identified with this method. As the mildly acidic sample processing conditions stabilize the succinimide intermediate. This suggests that the LERLIC MS/MS technique can be applied to proteome wide study of succinimide intermediates.
After its development, this technique paved the way for researchers in biochemistry to characterize protein deamidation in contexts ranging from archeology to biomedical research.
