Protamine sulfate precipitation method depletes abundant plant seed-storage proteins: A case study on legume plants

Depletion of abundant proteins is one of the effective ways to improve detection and identification of low‐abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable for their in‐depth proteome investigation. In this study, we provide evidence that the PSP method can also be effectively used for depletion of abundant seed‐storage proteins (SSPs) from the total seed proteins of diverse legume plants including soybean, broad bean, pea, wild soybean, and peanut. The 0.05% protamine sulfate (PS) was sufficient to deplete major SSPs from all legumes tested except for peanut where 0.1% PS was required. SDS‐PAGE, Western blotting and 2DE analyses of PS‐treated soybean and peanut seed proteins showed enriched spots in PS‐supernatant than total proteins. Coefficient of variation percentage (%CV) and principal component analysis of 2DE spots support the reproducibility, suitability, and efficacy of the PSP method for quantitative and comparative seed proteome analysis. MALDI‐TOF‐TOF successfully identified some protein spots from soybean and peanut. Hence, this simple, reproducible, economical PSP method has a broader application in depleting plant abundant proteins including SSPs in addition to RuBisCO, allowing discussion for comprehensive proteome establishment and parallel comparative studies in plants.