Plasma samples from 20 individuals were collected into identical EDTA-contain-ing tubes with a patented mechanical separator, either with or without the presence of a protease inhibitor cocktail. After collection, samples were processed as quickly as possible (within 15 min) into plasma, hence "time zero" analyses were from aliquots that were frozen at this earliest time point. Analysis was performed using H50 chips, as per manufacturer's instructions. Briefly, the chips are preincubated with 50% ACN, twice for 5 min each, followed by activation with binding buffer (10% ACN, 0.1 m NaCl, and 0.1% TFA) for 5 min and removed. Additional binding buffer (90 mL) is added, followed by 10 mL of plasma. After a 30 min incubation period, the chip is removed, and then washed twice by two 50 mL washes of binding buffer and another two washes with water. Surfaces are allowed to air dry, and matrix (1.5 mL of CHCA in 50% ACN, 0.1% TFA) is added, and allowed to air dry again prior to reading on a PBS2 instrument (Ciphergen Biosystems).
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