Identification of enzymes and activity from two-dimensional gel electrophoresis.
Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.
Afjehi-Sadat
Leila
L
Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria.
Lubec
Gert
G
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004798:Enzymes / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013116:Spinal Cord / Q000201:enzymology
D004798:Enzymes