생체시료 단백질 탈인산화 효소군의 정량적 질량분석기법 연구

Abstract

Phosphorylation is the major part of the signal transduction. Phosphorylation and dephosphorylation is strictly controlled and balanced by kinase and phosphatase, respectively. Though the amount of tyrosin phosphorylation is quite low, it has been intensively investigated. It is generally assumed that the level of Protein tyrosine phosphatases (PTPs) or active PTPs reflects cells status. There has been various attempts to quantify the PTP levels in the biological system. EGF treated HeLa cell system is chosen as model biological system to optimize PTP quantification. Since the abundance of PTPs is very low, they are hardly detected in the trypsin treated whole cell lysate. Since almost every PTPs have cysteine at their active site, enrichment by systein-capture was attempted. Biotin-PEO-iodoacetamide is utilized for capturing PTPs which has active site cysteine. The biotin parts of captured PTPs are reacted with Streptavidin beads. After washing and centrifugation, enriched proteins are digested with trypsin. Protein quantification is performed with triple quadrupole mass spectrometer (Waters, Quattro premiere) by Multiple Reaction monitoring (MRM) method. The specific tryptic peptides of PTPs are identified and they are heavy isotope labelled in advance. Since the amount of labelled peptides is known, the level of PTPs can be determined by comparing the area of same peptide sequence.