Matrix assisted laser desorption/ionization-time of flight-mass spectrometry analysis of proteins detected by anti-phosphotyrosine antibody on two-dimensional-gels of fibrolast cell lysates after tumor necrosis factor-alpha stimulation

We describe efficient methods for using functional proteomics analysis to s tudy signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-alpha. After stimulation with TNF-alpha, cellular proteins of L929 cells were extracted with a lysis buf fer containing 0.3% sodium dodecyl sulfate (SDS) for 10-30 min time interva ls, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosph atase-anti IgG antibody conjugate. To improve detection sensitivity by immu noblot analysis we used a chemifluorescent substrate for alkaline phosphata se. One hundred protein spots were detected in the TNF-alpha stimulated L92 9 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were ab le to detect time-dependent changes of a number of immunoblotted spots. Pro tein spots on a silver-stained 2-D gel corresponding to those detected by i mmunoblot analysis were subjected to in-gel trypsin digestion- matrix-assis ted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometr y analysis, respectively. Twenty-one proteins detected by immunoblot analys is were identified by MS-Fit database search analysis. Among them, the prot eins that shaw time-dependent changes in staining intensity include vimenti n, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromat in assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5 , and several other proteins. Vimentin and tubulin beta-chain have been rep orted to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-alpha. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of t ime-dependent changes in many proteins that are involved in signal transduc tion. Usefulness of the method for comprehensive analysis of the proteins i nvolved in signal transduction pathway and the limitations of the method ar e discussed.