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The Saccharomyces cerevisiae KNU5377 strain,which was isolated from spoilage in nature, has the ability toconvert biomass to alcohol at high temperatures and it canresist against various streses [18, 19]. In order to understandthe defense mechanisms of the KNU5377 strain under menadione(MD) as oxidative stress, we used several techniques forstudy: peptide mas fingerprinting (PMF) by matrix-asistedspectrometry (MS) followed by two-dimensional (2D) gelelectrophoresis, liquid chromatography electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS), and surface-enhanced laser desorption ionization-time of flight (SELDI-TOF)technology. Among the 35 proteins identified by MALDI-TOFMS, 19 proteins including Sod1p, Sod2p, Tsa1p, and Ahp1pwere induced under stress condition, while 16 proteins wereaugmented under normal condition. In particular, five proteins,Sod1p, Sod2p, Ahp1p, Rib3p, Yaf9p, and Mnt1p, were inducedin only stressed cells. By LC-ESI-MS/MS analysis, 37 proteinsthe stressed cells. Among the identified proteins, 32 proteins werefound in both cells. Five proteins including Yel047cp and Met6pwere only upregulated in the normal cells, whereas 17 proteinsincluding Abp1p and Sam1p were elevated in the stressed cells.It was interesting that highly hypothetical proteins such asYnl281wp, Ygr279cp, Ypl273wp, Ykl133cp, and Ykr074wpwere only expressed in the stressed cells. SELDI-TOF analysisusing the SAX2 and WCX2 chips showed that highly multiple-specific protein patterns were reproducibly detected in rangesTherefore, induction of antioxidant proteins, hypotheticalproteins, and low molecular weight proteins were revealed bydiferent proteomic techniques. These results suggest thatcomparative analyses using proteomics might contribute toelucidate the defense mechanisms of KNU5377 under MD stress.