Proteoformas de semillas de soja reveladas por proteómica basada en electroforesis bidimensional

Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations in resolving "proteoforms". Two-dimensional gel electrophoresis (2DE) has the potential to separate and visualize proteoforms effectively. To evaluate its utility, proteins extracted from soybean seed hypocotyls were analyzed by 2DE/LC-MS/MS. As a result, a total of 693 proteins were separated by 2DE, of which 302 were identified by LC-MS/MS. The dynamic range of protein abundance was approximately 10² to 10⁴. This analysis revealed that the hypocotyls contain numerous proteoforms of proteins essential for seed physiology, including late embryogenesis abundant protein, glycinin, β-conglycinin, trypsin inhibitor, sucrose-binding protein, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, 2DE revealed that the expression of proteoforms of the major seed storage proteins, glycinin A2 and A3 subunits, and β-conglycinin β subunit in hypocotyls differed from that in cotyledons, suggesting distinct functional roles beyond nutrient storage during germination. Overall, the results demonstrate that 2DE is a valuable complementary technique to shotgun proteomics, providing proteoform-specific information that cannot be resolved by shotgun analysis alone. SIGNIFICANCE: Shotgun proteomics is widely used for comprehensive profiling of protein expression. However, this approach has inherent limitations when analyzing "proteoforms". In the present study, we analyzed soybean seed hypocotyls using two-dimensional gel electrophoresis (2DE)-LC-MS/MS, demonstrating that 2DE is useful for effective proteoform analysis. 2DE provides proteoform-specific information that cannot be obtained from shotgun proteomics alone, making it a useful complementary method.