A reference map of human lung MRC-5 fibroblast proteins using immobilized pH gradient-isoelectric focusing-based two-dimensional electrophoresis

We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacr ylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a w ide range of investigations. The two-dimensional gel pattern of proteins ge nerated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particula r cell system provides a useful reference tool as a "master map" for subseq uent studies using those cells. In this map a total of 98 protein spots wer e identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionizati on time of flight following trypsin digestion. To increase the utility of t he reference map, cells were cultured in both Dulbecco's modified Eagle med ium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI) -1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. I n combination with the reference map, the standardised protocol described p rovides a tool for comparative studies involving MRC-5 cells in which nonsp ecific variation is minimized.