DEVELOPMENT OF NEW METHOD FOR ISOLATING C TERMINAL PROTEOLYTIC PEPTIDE

点击翻译按钮获取中文摘要

Isolation & MS characterization of the c-terminal peptide of proteins. Characterization of the C-terminus of a protein is important in identifying and confirming post-translational processing. Thus it becomes essential in quality control. One of the major limitations of the methodology available today is its sensitivity (usually 100-1000 picomoles of sample are required). In this project we develop a novel procedure that is both fast and sensitive (3). The strategy involves four steps: 1) The protein is degraded enzymatically 2) The c-terminal peptide is separated from all other proteolysis products through the use of anhydrotrypsin (a modified trypsin that binds only peptides containing K or R at the C-terminus) 3) The mass of the c-terminal peptide is determined by MALDI 4) The sequence of the c-terminal peptide is obtained by MS-MS. The procedure was tested and shown to work well on several proteins up to 50 KDa. However, there is no practical limitation in sequencing bigger proteins. The whole procedure takes about 30 minutes for each protein and several samples can be analyzed in parallel for the first three steps. Proteins present at the level as low as 10 picomoles were successfully sequenced.