Begin by normalizing the human tear protein samples to 50 micrograms of protein. Then add 200 millimolar dithiothreitol, or DTT, to a final concentration of 20 millimolar DTT, incubated at 95 degrees Celsius for 10 minutes. After cooling the protein solution to room temperature, add 400 millimolar iodoacetamide to a final concentration of 40 millimolar, then incubate in the dark at room temperature for 10 minutes.
Next, add 12%aqueous phosphoric acid in a one to 10 ratio, resulting in a 1.2%final concentration. Vortex the mixture briefly to mix the solution. Add protein binding buffer in a one to six ratio before vortexing again.
Uncap the suspension trapping micro spin column and assemble the spin column onto a two milliliter microcentrifuge tube to collect flowthrough. Add up to 200 microliters of acidified protein lysate mixture into the column. Centrifuge the column at 4000 G for 20 seconds.
Next, add 150 microliters of protein binding buffer and centrifuge as before to wash the suspended protein. Assemble the suspension trapping micro column onto a new 1.5 milliliter microcentrifuge tube. Carefully add 20 microliters TEAB digestion buffer onto the filter inside the spin column, avoiding bubbles and air gaps.
Securely cap the suspension trapping micro spin column to prevent evaporation. Incubate at 47 degrees Celsius for one hour in a thermal mixer without shaking. Elute peptides with 40 microliters of 50 millimolar TEAB.
Then centrifuge it at 4000 G for 20 seconds. Now add 40 microliters of 0.2%formic acid and centrifuge as before. Lastly, add 35 microliters of 0.2%formic acid and 50%to acetonitrile before centrifuging again.
Pull the three eluents and dry them using a vacuum centrifuge. Once dried, store the samples at 80 degrees Celsius for future analysis. The data dependent acquisition search yielded a total of 1183 plus or minus 118 proteins in the suspension trapping group, and 874 plus or minus 70 proteins at 1%FDR in the in-solution group.
Gene ontology analysis revealed similar proteomes for both approaches with main function as binding catalytic activity and molecular function regulation.
