Abstract
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.
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Acknowledgements
We are highly indebted to Professor Dr. Harald Höger (Institute for Animal Genetics, Medical University of Vienna) for supplying rat spinal cord tissues. We appreciate the technical assistance of Mrs. Maureen Cabatic-Felizardo and the general advice for use of enzyme protocols by Professor Dr. Christian Kubicek (Technical University of Vienna, Austria).
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Supplementary Table 1
Mass spectrometrical identification of metabolic enzymes in rat spinal cord tissue by MALDI-TOF/TOF analyses (PDF 23 kb)
Supplementary Table 2
Reagent and protocols for enzyme assays (PDF 129 kb)
Supplementary Table 3
Enzyme activities and underlying chemical reactions (PDF 22 kb)
Supplementary Table 4
Activity of metabolic enzymes identified in rat spinal cord tissue (PDF 16 kb)
Supplementary Table 5
Effects of inhibitors for malate dehydrogenase (PDF 7 kb)
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Afjehi-Sadat, L., Lubec, G. Identification of enzymes and activity from two-dimensional gel electrophoresis. Nat Protoc 2, 2318–2324 (2007). https://doi.org/10.1038/nprot.2007.317
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DOI: https://doi.org/10.1038/nprot.2007.317
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