Premium
Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cells
Author(s) -
Matsui Neil M.,
Smith Diana M.,
Clauser Karl R.,
Fichmann Jenny,
Andrews Lori E.,
Sullivan Christine M.,
Burlingame Alma L.,
Epstein Lois B.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150180315
Subject(s) - chromatography , cervical carcinoma , electrophoresis , chemistry , identification (biology) , gel electrophoresis , microbiology and biotechnology , cytokine , biology , biochemistry , immunology , cervical cancer , cancer , genetics , botany
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‐γ‐ (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.