Proteomeanalysisofmousebrain: Two-dimensionalelectrophoresisprofilesof tissueproteinsduringthecourseofaging Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hip- pocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was con- structed, which consists of the 2-DE map images and the respective spot data files with 15 related references. Keywords: Two-dimensional gel electrophoresis / Mouse brain / Aging / Protein identification / Two-dimensional electrophoresis database EL 3943 AkiraTsugita 1 TakaoKawakami 1* TatsuyaUchida 1 TomokoSakai 1 MasaharuKamo 1** TakuroMatsui 1 YuWatanabe 1 TadaomiMorimasa 2 KeiichiHosokawa 3 TosifusaToda 4 1 Research Institute for Biological Sciences, Science University of Tokyo, Yamazaki, Noda, Japan 2 Department of Neurology, Okayama University Medical School, Okayama, Japan 3 Institute of Bio- Microbiology, Minowa, Taito-ku, Tokyo, Japan 4 Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, Japan 1 Introduction The brain is situated in the center of the cerebrospinal nervous system in mammals. It is the center of all biologi- cal information receiving sensory signals and emitting motor signals to and from every part of the body. It func- tions in memory, thinking, emotion, and movement. It controls the behavior of animals and maintains physiologi- cal homeostasis. Brain consists of the forebrain or cere- brum, covered with cerebral cortex. The forebrain includ- es the basal ganglia, that is, thalamus, hippocampus, and amygdala. The lower structure includes the hypothala- mus, midbrain, brain stem, and cerebellum. Continuing from the brain stem is the spinal cord with cervical, thora- cic, lumbar, and sacral regions and peripheral nervous system. In humans a partial duplication of chromosome 21 results in brain-specific diseases, Down©s syndrome and sometimes Alzheimer©s disease. There are also addi- tional diseases of premature aging such as inherited Werner©s syndrome and Creutzfelt-Jacob©s syndrome, caused by the pathogenic protein ªprionº, Wilson©s dis- ease and a number of cerebrospinal disorders like amyo- trophic lateral sclerosis, Perkinsonism, etc. Addiction caused by a number of drugs such as opioid drugs, LSD, nicotine, alcohol, barbiturates, amphetamines, philopon, etc., is the result of drug abuse and affects the central nervous system. General proteome analysis aims at obtaining information that could not be furnished by mere DNA sequence analy- sis. By proteome analysis, in general, as well as separa- tion and identification of proteins electrophoretically sep- arated on 2-D gels [1], it is possible to learn, first, whether the individual genes in the genome of an organisms are expressed, in what type of cell, tissue and organ, and to what extent. Secondly, we learn when the gene is expressed in the life span of the organism, that is, whether the gene is expressed at early stages of develop- ment, starting with the fertilized egg to the embryo, fetus, and the newborn, or the following developmental stages, the young, mature and senescent period of life. Third, we Correspondence: Dr. Akira Tsugita, Proteomics Research Laboratory, 1-16-1 Amakubo Tsukuba, 305-0005 Japan E-mail: tsugita@proteo.gr.jp Fax: +81-298-51-8229 Abbreviations:BPB, bromophend blue; E-64, L-trans-epoxysuc- cinyl-leucylamido-(4-guanidino)-butane; TLCK, N-tosyl-L-lysyl- chloromethyl ketone Electrophoresis 2000, 21, 1853±1871 1853  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/0909-1853 $17.50+.50/0 * Current address: Nippon Glaxo, Tsukuba research Laborato- ries, 43 Wadai, Tsukuba, 300-4247, Japan ** Current address: School of Dentistry, Iwate Medical Univer- sity, 19-1 Uchimaru, Morioka, 020-8505, Japan Proteomicsand2-DE