IMMOBILIZED PH GRADIENT 2-DIMENSIONAL GEL-ELECTROPHORESIS AND MASS-SPECTROMETRIC IDENTIFICATION OF CYTOKINE-REGULATED PROTEINS IN ME-180 CERVICAL-CARCINOMA CELLS

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allo ws high resolution separation of proteins and their rapid identificati on. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a hi gh degree of reproducibility, good protein spot resolution, and a sele ction of pH range. Here we demonstrate the utility and efficacy of com bining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated prot eins in ME-180 cervical carcinoma cells. Three cytokine-regulated prot eins have been identified, using imidazole-zinc-stained preparative IP G 2-D gels and in-gel tryptic digestion followed by matrix-assisted la ser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, whi ch is important in protein degradation, and 3) Ran, a GTP-binding prot ein important in cell cycle regulation, protein import into the nucleu s, and RNA export from the nucleus.