Immobilized pH gradient two-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 allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser 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, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.
Matsui
N M
NM
Department of Pediatrics, University of California, San Francisco 94143, USA.
Smith
D M
DM
Clauser
K R
KR
Fichmann
J
J
Andrews
L E
LE
Sullivan
C M
CM
Burlingame
A L
AL
Epstein
L B
LB
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000494:pharmacology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis*; D046988:Proteasome Endopeptidase Complex; D014305:Triose-Phosphate Isomerase / Q000032:analysis*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D002583:Uterine Cervical Neoplasms; D020931:ran GTP-Binding Protein
D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020931:ran GTP-Binding Protein; D014305:Triose-Phosphate Isomerase