2.4 Protein Extraction From Frozen Tissue and FFPE Samples

To produce lysates for immuno-MRM analysis, frozen tissue was cryopulverized in a cryoPREP CP-02 (Covaris, Woburn, MA) and stored frozen until analysis. 5 μL of lysis buffer (25 mM Tris, 6 M Urea, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Sigma protease inhibitor (#P8340), 1% (v/v) Sigma phosphatase inhibitor cocktail 2 (#P5726), 1% (v/v) Sigma phosphatase inhibitor cocktail 3 (#P0044)) was added for each mg wet tissue weight (up to 1000 μL). The sample was vortexed for 10-15 sec and sonicated three times in a cup horn probe (filled with ice water) at 50% power for 30 seconds. The samples were stored in liquid nitrogen until the day of digestion.

FFPE samples were processed as described previously (48). Briefly, slide-mounted FFPE tissue sections were placed in a 4 slide holder. The slides were incubated three times in xylene for 3 min followed by 100% (v/v) ethanol twice for 3 min. The tissue was then hydrated twice in 85% (v/v) ethanol for 3 min, 70% (v/v) ethanol for 3 min, and distilled water for 3 min. The tissue was then blotted and scraped off the slide into a screw cap microfuge tube. To each sample tube (containing three FFPE 10 µm tissue sections), extraction buffer (0.2% RapiGest in 50 mM ammonium bicarbonate, NH₄HCO₃) was added and incubated at 95°C for 30 minutes with mixing at 1000 rpm (Thermomixer, Eppendorf, Enfield, CT). The samples were then cooled on ice for 5 minutes and sonicated twice in a cup horn probe (filled with ice water) at 50% power for 30 sec. The samples were then incubated at 80°C for 120 minutes with mixing at 1000 rpm and then cooled on ice for 5 min. 100 μL of 50 mM NH₄HCO₃, pH 8.0 was added, and the samples were sonicated twice in the cup horn probe (filled with ice water) at 50% power for 30 sec. Following processing, all samples were stored at -80°C until the day of digestion.

Protein concentrations of lysates were measured in triplicate using Micro BCA Protein Assay Kit (Pierce, #23235).