Identification of polysaccharide binding proteins by affinity electrophoresis in inhomogeneous polyacrylamide gels and subsequent SDS-PAGE/matrix-assisted laser desorption ionization-time of flight analysis.
A procedure that allows the identification of polysaccharide binding polypeptides is described. The method can be applied to proteins whose enzymatic activity is either unknown or cannot be identified unambiguously by activity-staining procedures and it has been used for very complex protein mixtures, such as crude extracts of plant organs. The procedure consists of three steps. First, an affinity polyacrylamide gel electrophoresis using an inhomogeneous polyacrylamide slab gel composed of two triangular parts, an upper gel lacking the ligand and a lower triangular gel containing an immobilized ligand, is performed. Proteins that interact with the ligand form bands that deviate from those of nonbinding proteins and can be detected by protein staining (or, if possible, by activity staining). Second, the bands containing the interacting proteins are excised, denatured, and subjected to SDS-PAGE using a slab gel. In the resulting protein pattern the target proteins cover most of the length of the gel piece applied to the SDS gel, whereas contaminating proteins appear as spots or narrow bands. Suitable regions of the target protein bands are selected for tryptic digestion. Third, the resulting peptides are analyzed by matrix-assisted laser desorption ionization-mass spectrometry followed by database research.
Eckermann
Nora
N
Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Haus 20, D-14467 Golm, Germany.
Fettke
Jörg
J
Steup
Martin
M
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D006005:Phosphorylases / Q000032:analysis / Q000378:metabolism; D010936:Plant Extracts / Q000032:analysis / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry
D007527:Isoenzymes; D010936:Plant Extracts; D010940:Plant Proteins; D011134:Polysaccharides; D006005:Phosphorylases; D014357:Trypsin