ium 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, San Jose, CA, USA). Ion optics were tuned using Angiotensin at 500 fmol/μL [50% (v/v) methanol, 1.0% (v/v) formic acid], and capillary temperature step optimized for peptide desolvation under normal LC workflows with purified BSA peptides (Michrom Bioresources, Inc., Auburn, CA, USA) using the gradient described below. Sample handling and LC separations for LC-SRM analyses were all performed using an Eksigent nanoLC ultra 1D plus [0.1% (v/v) formic acid in MilliQ water (18.2 MΩ) as solvent A, and 99.9% (v/v) ACN, 0.1% (v/v) formic acid as solvent B]. Peptides were trapped and washed on a C8 Cap Trap (Michrom Bioresources, Inc., Auburn, CA, USA) using solvent A at 5 μL/min for 2 min. Peptide chromatography used a 25 min non-linear gradient from 2 to 60% over 12.5 min, a washing step, and a 9 min equilibration step at 2% solvent B all using a flow rate of 500 nL/min. AQUA™ peptides used were designed and described in Houston et al. (2010). All are listed in Table A1 in Appendix. Precursor and product ions, ion types, and charge states are also listed in Table A2 in Appendix. The development of an optimal LC-MRM analysis, including most abundant precursor and product ion selection and collision energy optimization, was aided by iterative analysis of soybean tryptic digests using Pinpoint 1.1 (Thermo Fisher Scientific, 2011). Peptide sequences were entered into Pinpoint manually and the recommended workflows for precursor/product ion selection were followed. When necessary, precursor and product ions were chosen manually by direct infusion of peptides at 500 fmol/μL in 50% (v/v) ACN, 1.0% (v/v) formic acid using product ion scans from the most intense precursor. At least five transitions were chosen for each peptide. Cycle times and chromatography were optimized to ensure 10 measurements for each transition across the chromatographic peak profile.
Selected Reaction Monitoring results were provided by LCQuan 2.6 (Thermo Fisher Scientific, 2009). Peak detection and integration were performed using the ICIS algorithm wit