STRATEGIES FOR NONRADIOACTIVE METHODS IN THE LOCALIZATION OF PHOSPHORYLATED AMINO-ACIDS IN PROTEINS

被引:29
作者
MEYER, HE
EISERMANN, B
HEBER, M
HOFFMANNPOSORSKE, E
KORTE, H
WEIGT, C
WEGNER, A
HUTTON, T
DONELLADEANA, A
PERICH, JW
机构
[1] FISONS INSTRUMENTS VG BIOTECH, ALTRINCHAM WA14 5RZ, CHESHIRE, ENGLAND
[2] UNIV PADUA, DIPARTIMENTO CHIM BIOL, I-35121 PADUA, ITALY
[3] UNIV MELBOURNE, SCH CHEM, PARKVILLE, VIC 3052, AUSTRALIA
关键词
PHOSPHOTYROSINE; PHOSPHOSERINE; PHOSPHOPROTEIN; PHOSPHOTHREOUINE; MASS SPECTROMETRY; SEQUENCE ANALYSIS;
D O I
10.1096/fasebj.7.9.7687226
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [P-32]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.
引用
收藏
页码:776 / 782
页数:7
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