A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis.
Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.
Song
Jun
J
Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia B4N 1J5, Canada. songj@agr.gc.ca
Braun
Gordon
G
Bevis
Eric
E
Doncaster
Kristen
K
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D005638:Fruit / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000302:isolation & purification*
D010940:Plant Proteins