Prior to microSol IEF, varying amounts of serum or plasma up to 250 mL, with or without protein depletion, were diluted to 1 mL in 8 m urea, 10 mM glycine. Samples were reduced with 10 mM DTT for 1 h at RT, concentrated to 200 mL using a 5K MWCO spin concentrator, rediluted to 1 mL with 8 m urea, 10 mM glycine (to dilute reducing reagents), and alkylated with 25 mM iodoacetamide (IAM) for 2 h at RT in the dark. Alkylation was quenched with 1% DTT for 15 min at RT. Following insolution reduction and alkylation, depleted plasma or serum samples were pre-fractionated by microscale solution IEF as previously described , using a ZOOM-IEF fractionator (Invitrogen, Carlsbad, CA, USA). Proteins were separated into seven small volume (~700 mL) pools where the separation chambers were defined by immobilized gel membranes having pH values of 3.0, 4.4, 4.9, 5.4, 5.9, 6.4, 8.1, and 10.0, respectively. In these experiments the pH 3.0 and 10.0 membranes were commercially available (Invitrogen), while the remaining membranes were prepared as described previously .
For protein array pixelation, ZOOM-IEF fractions were further separated by 1-D PAGE in individual lanes on short minigels. In general, the highest possible protein amounts that did not cause extensive band distortion were loaded into 10-well 10% NuPAGE™ (Invitrogen) SDS gels and electrophoresed using MOPS running buffer until the tracking dye had migrated 4 cm. Proteins were visualized by staining with Colloidal Blue (Invitrogen). Each lane was subsequently cut into uniform 1 mm slices with the MEF-1.5 Gel Cutter (The Gel Company, San Francisco, CA, USA). Two gel slices were combined per digestion tube and the gel slices were in-gel trypsin-digested as previously described , and analyzed by LC-MS/MS.
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