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-a stimulation We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-a. After stimulation with TNF-a, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10±30 min time intervals, and were separated by two-dimensional (2-D) electrophore- sis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immuno- blot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hun- dred protein spots were detected in the TNF-a stimulated L929 cell extract by immuno- blot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel correspond- ing to those detected by immunoblot analysis were subjected to in-gel trypsin diges- tion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysiswereidentifiedbyMS-Fitdatabasesearchanalysis.Amongthem,theproteins that show time-dependent changes in staining intensity include vimentin, tubulin beta- chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 sub- unit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-a. 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 time- dependent changes in many proteins that are involved in signal transduction. Useful- ness of the method for comprehensive analysis of the proteins involved in signal trans- duction pathway and the limitations of the method are discussed. Keywords: Functional proteomics / Signal transduction EL3954 Mitsuaki Yanagida 1,2 Yutaka Miura 1 Kazumi Yagasaki 1 Masato Taoka 3 Toshiaki Isobe 3 Nobuhiro Takahashi 1 1 Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo, Japan 2 New Energy and Industrial Technology Development Organization, Tokyo, Japan 3 Laboratory of Biochemistry, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan 1 Introduction Tumor necrosis factor (TNF) is a pleiotropic cytokine. Its varied biological activities are signaled through two dis- tinct cell surface receptors, CD120a (TNFR1) and CD120b (TNFR2), which are expressed on most cell types [1]. When activated by TNF-a, these two receptors generate largely nonoverlapping signals. The majority of the known activities of TNF-a, which include the pro- grammed cell death known as apoptosis, antiviral activity in a variety of cell types, and tumor promoter activity on cancer cells [2], has been attributed to CD120a (TNFR1). On the other hand, CD120b (TNFR2) is involved in necrotic effects of TNF-a. The mice lacking this receptor showed a dramatic decrease in tissue necrosis upon TNF-a injection [3]. Thus, TNF-a triggers a biochemical pathway that leads to the apoptosis or necrosis, depend- ing on receptors with which it interacts. However, it also activates a pathway that blocks this very pathway, and so sets up a delicate life-death balance within the cell [4]. A number of molecules that participated in the death signal- ing pathway have been identified; these include TNFR1- associated death domain protein (TRADD), Fas-associat- ing protein with a novel death domain (Mort-1/FADD), Bad, and caspases. Meanwhile, the molecules that par- ticipated in the survival signaling pathway are receptor Correspondence: Dr. Mitsuaki Yanagida, Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan E-mail: myanagid@cc.tuat.ac.jp Fax: +81-42-367-5709 Abbreviations: IL, interleukin; TNF-a,tumornecrosisfactor-a 1890 Electrophoresis 2000, 21, 1890±1898  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/0909-1890 $17.50+.50/0