Following MicroSol-IEF, the fractions were separated by 1-D PAGE in individual lanes on short minigels. In some cases, proteins were extracted from the membrane partitions by two sequential incubations with 400 mL of MicroSol buffer. Each fraction was run on separate gels to avoid possible cross-contamination from other fractions. The highest possible protein amounts that did not cause extensive band distortion were loaded into 10-well 10% NuPAGE (Invitrogen) SDS gels and elec-trophoresed using MOPS running buffer until the tracking dye had migrated 4 cm (plasma analysis) or 6 cm (serum analysis) into the gels. In fractions containing very low amounts of proteins (F1 and M1 of serum analysis), the proteins were concentrated by precipitation with 9 vol of acetone and electrophoresed for only 2 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). Generally, two gel slices were combined (i.e., 2 mm pixels) per digestion tube and the pixels were digested in-gel with trypsin as previously described , and analyzed by LC-ESI-MS/MS. In the serum analysis, the gel lanes were pixelated in a variable manner depending on the band intensity. Intense bands were digested as 1 mm pixels, while regions of the lane without much staining were digested as 4 mm pixels.
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