The formalin-fixed, paraffin-embedded (FFPE) tissues and TMA were prepared as previously described.[19 (link)] Briefly, tissues were heated, dewaxed, antigen retrieved at pH 9, and sprayed with a thin molecular layer of collagenase type III (COLase3; StemCell Technologies) using an automated sprayer (M3 TM-Sprayer, HTXImaging, Chapel Hill, NC, USA). COLAse3 was sprayed onto tissues with the same automated sprayer using parameters of 45°C, 10 psi, 25 μL/min, 1200 velocity, and 15 passes with a 3.0 mm offset. Samples were digested in high humidity at 37.5°C for five hours followed automated spraying of CHCA matrix prepared as 7 mg/mL in 50% acetonitrile, 1% TFA with a spiked standard of 200 femtomole/microliter [Glu1]-Fibrinopeptide B human (Glufib) (Sigma-Aldrich), St. Louis, MO, USA). CHCA was sprayed onto tissues with the same automated sprayer using parameters of 77°C, 10 psi, 100 μL/min, 1300 velocity, and 10 passes with a 2.5 mm offset.