Sample processing for quantitative proteomics
Extraction and in-solution trypsin digestion of proteins were performed as previously reported (Kültz, Li, Gardell, & Sacchi, 2013) with the following changes: After transfer of crushed sample powder to a low retention Eppendorf tube (LR-MCF), proteins were dissolved in 8M urea at 1.5x sample wt/vol and reduced with 10mM dithiothreitol (DTT) for 30 min. at 37oC. Proteins were then alkylated with 30 mM iodoacetamide (IAA) at RT for 30 min in the dark. Proteins were precipitated in an ice-cold solution of 10% trichloroacetic acid (TCA)/90% Acetone/0.15% DTT (5x the total volume). Samples were then centrifuged at 20,000 g for 5 min (4 °C) and the precipitated protein pellet washed once in 100% acetone/0.15% DTT. The protein pellet was re-dissolved in 8M urea (5x the original tissue wt/vol) for 30 min at RT on a rotator. After centrifugation at 19,000 g (5 min,) the supernatant was transferred to a clean LR-MCF tube and stored at 4 °C. Duplicate aliquots were used to determine protein concentration using a BCA protein assay compatible with diluted urea (Thermo-Pierce, cat. 23225). The appropriate amounts of LCMS grade water and 1M ammonium bicarbonate (Ambic) buffer (pH 8.5, final concentration 100 mM) added to dilute samples to 150 ng/100 µl total protein concentration in 0.6 ml LR-MCF tubes. Immobilized trypsin (Promega cat. V9012) was added at a 1:25 ratio relative to total protein and the samples incubated in a rotator at 35 °C for exactly 16h. Trypsin beads were removed by centrifugation for 2 min at 500 g and supernatant was transferred to a clean 0.6 ml LR-MCF tube. Any remaining undissolved solids were removed by centrifugation for 5 min at 19,000 g and supernatant was transferred to another clean 0.6 ml LR-MCF tube. Samples were then dried by speedvac (Thermo-Savant, ISS-110) until urea precipitate began forming. Peptides where then resuspended in 150 µl LCMS grade water containing 0.1% formic acid (FA) and transferred to total recovery glass vials (Waters 186000384C) for sample injection.
Peptide samples (2 µl, 100 ng/µl) were injected with a nanoAcquity sample manager (Waters, Milford, MA), trapped for 1 min at 15 µL/min on a Symmetry trap column (Waters 186003514), and separated on a 1.7µm particle size BEH C18 column (250mm x 75µm, Waters 186003545) by reversed phase liquid chromatography using a nanoAcquity binary solvent manager (Waters). Peptides were eluted using a 125 min linear gradient ranging from 3% to 35% acetonitrile (ACN) directly (online) into a UHR-qTOF mass spectrometer (Impact II, Bruker) using a pico-emitter tip (New Objective FS360–20-10-D-20, Woburn, MA). Batch processing of samples was controlled with Hystar 4.1 (Bruker) and a quality control standard (68 fmol BSA peptide mix) was used at least once a week to monitor instrument performance.