Premium
Matrix‐assisted laser desorption mass spectrometric peptide mapping of proteins separated by two‐dimensional gel electrophoresis: Determination of phosphorylation in synapsin I
Author(s) -
Zhang Wenzhu,
Czernik Andrew J.,
Chait Brian T.,
Yungwirth Tom,
Aebersold Ruedi
Publication year - 1994
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560030415
Subject(s) - peptide , chemistry , cyanogen bromide , chromatography , gel electrophoresis , membrane , phosphorylation , mass spectrometry , alamethicin , matrix assisted laser desorption/ionization , membrane protein , biochemistry , peptide sequence , desorption , lipid bilayer , organic chemistry , adsorption , gene
A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2‐dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys‐C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix‐assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (± 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2‐dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca 2+ /calmodulin‐dependent protein kinases, which appear to control their biological activity.