一种有效检测水稻叶鞘低丰度蛋白的方法

蛋白质组学研究中,低丰度蛋白的富集、检测和鉴定是主要的技术难题。植物组织中核酮糖-1,5-二磷酸羧化/加氧酶(rubisco)等高丰度蛋白占细胞全蛋白很大比例,严重影响双向凝胶电泳(2-DE)对低丰度蛋白的检测。为探索一种适用于叶鞘低丰度蛋白的有效检测方法,本研究采用聚乙二醇(PEG)沉淀法以减少rubisco等高丰度蛋白,富集低丰度蛋白。以Mg/NP-40法为对照,分别使用15%和20%PEG分离水稻叶鞘可溶性全蛋白,并将对照和各PEG分离组分进行2-DE。2-DE图谱经软件分析结果表明,20%PEG沉淀法适用于灌浆期叶鞘低丰度蛋白检测。

An approach to detect the low-abundant proteins in rice leaf sheath

Enrichment of low-abundant proteins and their detection still remain great challenges in proteomics research.The existence of high-abundant proteins,e.g.ribulose-1,5-bisphosphate carboxylase/oxygenase(rubisco) in plants,leads to this dilemma.The high-abundant proteins engage a large proportion of the whole-cell proteins and thus make the low-abundant proteins poorly detectable by 2-DE.In this report,we used a protocol for the preparation of whole-cell proteins through polyethylene glycol(PEG) precipitation,to develop an approach of identifying low-abundant proteins in rice leaf sheathes.In a comparison of the 2-DE analyses of protein samples prepared using the Mg/NP-40 method without PEG fractionation,with the 15% and 20% PEG fractionation protocol,a relatively high reproducibility was achieved using the 20% PEG fractionation protocol in terms of protein species and low-abundant proteins.