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Characterization of rat brain stathmin isoforms by two‐dimensional gel electrophoresis‐matrix assisted laser desorption/ionization and electrospray ionizationion trap mass spectrometry
Author(s) -
Zugaro Lisa M.,
Reid Gavin E.,
Ji Hong,
Eddes James S.,
Murphy Anne C.,
Burgess Antony W.,
Simpson Richard J.
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150190544
Subject(s) - stathmin , phosphorylation , chemistry , gene isoform , isoelectric point , mass spectrometry , kinase , electrospray ionization , protein kinase a , phosphoprotein , phosphopeptide , peptide mass fingerprinting , microbiology and biotechnology , biochemistry , proteomics , biology , chromatography , enzyme , gene
Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor‐regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two‐dimensional electrophoresis (2‐DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen‐activated protein (MAP), cdc2 kinase, protein kinase A, and Ca 2+ /calmodulin‐dependent kinase‐Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post‐translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2‐DE with apparent molecular weight ( M r )/isoelectric point (p I ) values of 15 500/6.2, 15 000/6.1, and 15 000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix‐assisted laser desorption/ionization mass spectrometry and electrospray‐ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the M r /p I locus of this region of the 2‐DE gel map. These include: phosphatidyl ethanolamine binding protein ( M r ∼18 000 /p I 6.0), synuclein forms 2 and 3 ( M r ∼14 000 /p I 5.4), and synuclein form 2 ( M r ∼15 000 /p I 5.0).