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제105회 대한화학회 학술발표회, 총회 및 기기전시회 안내 Application of Isotope_Coded N-terminal Sulfonation to Study Ubiqitinated Peptides

등록일
2010년 2월 24일 17시 35분 07초
접수번호
1339
발표코드
34P205포 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
발표시간
목 <발표Ⅱ>
발표형식
포스터
발표분야
물리화학
저자 및
공동저자
장문석, 김인애, 이상원
고려대학교 화학과, Korea
Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Here we report the development of a novel strategy for searching ubiquitination site based on the N-terminal sulfonation. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated Gly-Gly branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. We present here a novel stable-isotope labeling method to simultaneously identify and quantify ubiquitin conjugated proteins produces Gly-Gly branched peptides by specifically derivatizing the N-terminus of proteins with 4-sulfophenyl isothiocyanate (SPITC). We describe the isotope coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo sequencing to be studied simultaneously. The experimental and ubiquitinated peptide mixture were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled Gly-Gly branched peptides in which each isotopic pair differs in mass by 12Da. The 13C-labelled SPITC resulted inconvenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by LC-MS/MS. Our results show that the LC-MS/MS analysis of sulfonated peptides can provide a highly effective method for the determination of ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.

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