C.
Twenty-five microliters of precipitated protein was collected by centrifugation at 16,000 × g at 4°C for 1 min. Pellets were dissolved in 300 μL of urea buffer (50 mM Tris-HCl, pH 8.0, and 8 M urea) by pipetting and vortexing, and finally centrifuged at 16,000 × g at 4°C for 10 min to pool and pellet insoluble material. Supernatants were collected, split into two equal portions and snap frozen in liquid nitrogen and stored at −80°C. Dissolved protein samples were thawed at 4°C, and vortexed to make homogenous. Samples were quantified in quadruplicate using 3 μL of undiluted sample per replicate. Quantification was accomplished using the Coomassie dye-binding assay at 1× (Bio-Rad, Hercules, CA, USA) using the 96-well plate format employing an in-house-prepared soy protein (Williams 82) standard at 2 mg/mL in urea buffer. A five point standard curve of 0 (3 μL of urea buffer), 1, 2, 4, and 8 mg/well was created using a twofold dilution series made with urea buffer. The standard curve used 4 μL/well, and was done in triplicate. Final protein concentration was the average of the protein concentrations obtained from the four replicate assays. After the concentrations were determined, the reconstitution step was repeated using back-calculated values for volume (using between 200 and 800 μL/sample) in order to reconstitute each sample to a concentration of 1–2 mg/mL. Protein quantification was repeated in the same manner, using one of the frozen aliquots, to verify the final concentrations were between 1 and 2 mg/mL.
The remaining aliquot of frozen protein was thawed at 4°C, vortexed to make homogenous and 10 μg of each sample was portioned into 1.5 mL polypropylene tubes. For each, the volume was brought to 10.0 μL using 8 M urea buffer. Disulfide bonds were reduced with 15.0 μL of reduction solution (16.7 mM dithiothreitol (DTT), 6.7 ng/μL BSA in 50 mM ammonium bicarbonate) for 1 h at room temperature. Reduced cysteines were carboxyamidomethylated with 5.0 μL alkylation solution (300 mM iodoacetamide in 50 mM ammonium bicarbonate) at room temperature in the dark for 1 h. Urea was diluted to 0.67 M and iodoacetamide was neutralized with equimolar DTT by adding 90 μL of neutralization solution (16.7 mM DTT in 50 mM ammonium bicarbonate) and incubating at room temperature for 15 min. Samples were chilled to 4°C and 10.0 μL of cold trypsin solution (20 ng/μL sequencing grade – modified, Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate) added. Digestion was allowed to proceed for 16 h at 37°C. AQUA™ peptides (Sigma Life Science, The Woodlands, TX, USA) in 50% acetonitrile (ACN), 1.0% (v/v) formic acid were added to the completed digests, which were frozen and evaporated to dryness using a CentriVap before being stored at −80°C.
AQUA™ peptides were synthesized by Sigma-Aldrich Co. LLC., to greater than 95% purity, and were portioned in 0.5 nmol aliquots and stored lyophilized at −20°C until use. AQUA™ peptides were dissolved to 2.5 pmol/μL using 50% (v/v) ACN (Sigma-Aldrich, Saint Louis, MO, USA), 1.0% (v/v) formic acid (Acros Organics, New Jersey, USA) with 5 × 10 s of vortexing max speed. Portions of 100 pmol were made by aliquoting 40 μL to separate tubes and storing at −80°C after centrifugal evaporation. Prior to use, peptide aliquots were dissolved in 50% (v/v) ACN, 1.0% (v/v) formic acid in an appropriate volume so that when mixed together, the final AQUA™ concentration is 50 fmol/μL (except for glycinin G1, which was at 100 fmol/μL). AQUA™ peptides at these concentrations were added to completed digests at 2 μL/μg of digested material.
All sample digests were dissolved to 200 ng/μL with 5% (v/v) ACN, 0.1% (v/v) formic acid with vortexing (3 × 10 s max speed). Insoluble debris was pelleted with centrifugation at 21,000 × g for 2 min at room temperature. Clearspun samples were immediately transferred to a 96-well plate, covered with film and placed in the 8°C autosampler tray. All mass spectrometry was performed using a TSQ Vantage Extended Mass Range triple quadrupole mass spectrometer (Thermo Fisher Scientific, S