Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis.
A modified phenol-based protocol and a phenol-free protocol that involves hot SDS extraction followed by TCA precipitation in acetone were qualitatively and quantitatively compared and evaluated on apple peel and strawberry fruit. The phenol protocol resulted in significantly higher protein yields of 2.35 +/- 0.1 and 0.46 +/- 0.06 mg/g of FW from apple and strawberry fruit, respectively, compared to the SDS protocol, which produced 0.74 +/- 0.1 and 0.27 +/- 0.02 mg/g of FW, respectively. 2-DE analysis of apple protein extracts revealed 1422 protein spots associated with the phenol protocol and 849 spots associated with the SDS protocol. Of these, 761 were present only in phenol gels, whereas 23 were exclusive to SDS samples. For strawberry, SDS extraction produced poor-quality spots with a high degree of streaking, indicating possible contamination. The application of a cleanup procedure resulted in a purified protein extract with high-quality spots. 2-DE analysis of strawberry protein extracts revealed 1368 spots for the phenol protocol and 956 spots for the SDS protocol accompanied by the cleanup procedure. Of these, 599 spots were present only in phenol gels, whereas 109 were present only in SDS samples. Spots from each fruit tissue and extraction procedure were selected, and a total of 26 were identified by LC-MS/MS. Overall, this study demonstrates the complexity of protein extraction of fruit tissues and suggests that a phenol-based protein extraction protocol should be used as a standard procedure for recalcitrant fruit tissues, whereas a SDS protocol with or without a cleanup procedure may be used as an alternative protocol.
Zheng
Qifa
Q
Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main Street, Kentville, Nova Scotia B4N 1J5, Canada.
Song
Jun
J
Doncaster
Kristen
K
Rowland
Elden
E
Byers
David M
DM
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D031985:Fragaria / Q000737:chemistry*; D005638:Fruit / Q000737:chemistry*; D027845:Malus / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D019800:Phenol; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate
D010940:Plant Proteins; D019800:Phenol; D012967:Sodium Dodecyl Sulfate