Isolation and Discovery of Small Peptides as Potential Biomarkers in Three Rice Cultivars Using Surface-Enhanced Laser Desorption/Ionization (SELDI) Time-of-Flight (TOF) Mass Spectrometry (MS)

Low molecular weight proteins are promising molecules for rice plants. The objectives of this study were to develop an optimized method for protein extractions and protein chips and to perform practical application for isolation of low molecular weight proteins/peptides as potential biomarkers of three rice cultivars, Ilpumbyeo, Hwaseongbyeo, and Nakdongbyeo, using surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS). Among five protein extraction methods tested, we found that the trichloroacetic acid (TCA)-acetone method was the most effective for the extraction of low molecular weight proteins/peptides. Four protein chip arrays, including CM10 (weak cation exchanger), Q10 (strong anion exchanger), H50 (reverse phage), and IMAC30 (immobilized metal affinity), were used under low and high stringency conditions at varying pH with two energy absorbing molecules, alpha-Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA). Of the 70 proteins isolated by the Q10 array, 20 showed significant differences in expression between the three cultivars (p < 0.05). Principal component analyses showed distinct expression patterns in the three cultivars, which were confirmed by hierarchical cluster analysis-based heat mapping. Significantly upregulated proteins were divided into five cultivar-specific clusters by heat mapping. Taken together, the results indicate that rice cultivar-specific small peptides that may be the potential biomarkers have been proposed by the TCA-acetone extraction method in combination with the Q10-SA chip array.