Sample preparation focusing on plant proteomics: extraction, evaluation and identification of proteins from sunflower seeds

Abstract

In the present work 17 different procedures for extraction of proteins from sunflower (Helianthus annuus L.) seeds in natura were evaluated regarding extraction time and temperature, type of solvent, and analytical procedure. After each extraction procedure, total protein was determined, ranging from 260 ± 5 to 2727 ± 11 μg g⁻¹. Then, a 2-D PAGE was used for P4, P5, P13 and P17, which were considered the most efficient extraction protocols, to calculate the match between them. The highest (77 ± 3%) and the lowest (48 ± 1%) values were achieved when P13 × P17 and P4 × P17 protocols were compared, respectively. Additionally, the P4 and P5 protocols presented the highest number of spots (196 ± 11 and 194 ± 20) after 2-D PAGE protein separation. From both protocols (P4 and P5), 66 protein spots were visualized in both extraction protocols and also analyzed by the 2-D PAGE technique for verifying possible changes in protein expression. Thirty-six spots were differentially found at 90% (or 1.8 times) and related to their relative volume and/or intensity. From these protein spots, 24 (ca. 67%) were successfully identified, 17 being proteins from sunflower seeds. The others are homologous proteins from other plant organisms. All of these analyses contributed to choosing the protocol P4 as the best for protein extraction from sunflower seeds.