title: Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method.
abstract: More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B(act), and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B(act), and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.
authors.affiliation: Dept. of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Dmitry E
authors.initials: DE
authors.lastname: Agafonov
authors.forename: Jochen
authors.initials: J
authors.lastname: Deckert
authors.forename: Elmar
authors.initials: E
authors.lastname: Wolf
authors.forename: Peter
authors.initials: P
authors.lastname: Odenwälder
authors.forename: Sergey
authors.initials: S
authors.lastname: Bessonov
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics*
chemical_list: D011506:Proteins