去除紫花苜蓿叶片高丰度蛋白的方法及其应用

紫花苜蓿叶片中大量的高丰度蛋白(核酮糖-l,5-二磷酸羧化/加氧酶,Rubisco)干扰了蛋白质的动态分辨率,严重影响蛋白质组学研究中功能蛋白的检测与鉴定。为了探究去除高丰度蛋白的适宜方法,本研究利用Mg/NP-40与聚乙二醇(PEG)预分离紫花苜蓿叶片蛋白,通过双向凝胶电泳法比较了不同浓度PEG对叶片高丰度蛋白的分离情况。电泳图谱显示0,15%,17.5%,20%PEG处理的蛋白质中分别可以检测到(335±17),(417±3),(445±7),(459±11)个蛋白质点,0,15%,17.5%处理组间差异显著(P<0.05),17.5%和20%PEG 处理组间没有差异(P<0.05)。然而,17.5%PEG能够检测到更多的差异蛋白质点,证明其更能有效沉淀高丰度蛋白,便于检测被Rubisco遮盖的蛋白质点。将该方法应用于紫花苜蓿叶片响应低温胁迫的蛋白质组学研究中检验其应用效果,与三氯乙酸/丙酮法提取的全蛋白相比,去除高丰度蛋白后鉴定出8个新的蛋白质差异点,证明该方法适用于实际的蛋白质组学研究。可见,Mg/NP-40与17.5%PEG法是最适宜去除紫花苜蓿叶片高丰度蛋白的方法。

Proteomic methods for removing high-abundance proteins in alfalfa leaf

The higher content of high-abundance proteins (Ribulose-l,5-bisphosphate carboxylase/oxygenase;RuBisCo)interferes with the dynamic resolution of proteins in two-dimensional electrophoresis (2-DE),affect-ing the detection and identification of functional proteins in proteomics.To explore suitable methods for remo-ving high-abundance protein,Mg/NP-40 and different concentrations of polyethylene glycol (PEG)were used for protein pre-fractionation and the treatments’influence on the separation of proteins in alfalfa leaf was com-pared.The results indicated that (335±17),(417±3),(445±7)and (459±11)spots were detected in treat-ments of 0,15%,17.5% and 20% PEG respectively.There were significant differences between the 0 -17.5% treatments but no significant differences were found between the 17.5% and 20% treatments.More differential protein spots were detected in the 17.5% treatment,which thus proved the most effective way of removing RuBisCo proteins.In order to inspect the applicability of this result,a proteomic study of alfalfa in response to low temperatures was under taken.Compared with total proteins extracted by TCA/acetone,eight new protein spots were identified after PEG treatment to remove high-abundance proteins.The research thus indicates that pre-fractionation with Mg/NP-40 and17.5% PEG is suitable for proteomic studies of alfalfa.