基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法

Absolute Quantification of Proteins in Rice Leaf Based on Multiple Reaction Monitoring Mass Spectrometry

【目的】建立水稻叶片蛋白的多反应监测(MRM)质谱绝对定量方法。【方法】水稻叶片蛋白经含1.0%十二烷基硫酸钠(SDS)的磷酸盐(PBS)缓冲液提取,丙酮沉淀除杂纯化、胰蛋白酶消化,酶解液经液相色谱分离,MRM质谱监测,外标法定量。【结果】向提取缓冲液中加入1.0%SDS可增强水稻叶片中16种靶蛋白和总蛋白质的提取效果;不同有机试剂处理,总蛋白质沉淀量存在显著差异(P<0.05),沉淀能力从强到弱依次为乙腈>丙酮>异丙醇>甲醇>乙醇;对于16种目标蛋白,总体以丙酮沉淀效果最好,其次是异丙醇和乙腈,甲醇和乙醇效果较差。该方法线性范围均达到3个数量级,定量限为0.1~2.5 nmol/L,灌浆期16种水稻叶片蛋白质含量为6.0~2818.1μg/g,相对标准偏差均小于14%。【结论】SDS可显著提高水稻叶片蛋白提取效果,采用丙酮或乙腈可获得较好的蛋白沉淀效果,但不同蛋白质略有差异。结合MRM质谱监测技术可实现水稻叶片蛋白的绝对定量,方法线性范围宽、敏度高、重复性好。

【Objective】An absolute quantitative method for proteins in rice leaf based on multiple reaction monitoring(MRM)mass spectrometry was established.【Method】The proteins in rice leaf were extracted with PBS buffer containing 1.0%sodium dodecyl sulfate(SDS),purified by acetone precipitation,and then digested by Trypsin.The enzymatic hydrolysate was separated by liquid chromatography,monitored by MRM mass spectrometry and quantified by external standard method.【Results】The addition of 1.0%SDS to the extraction buffer enhanced the extraction effect of 16 kinds of target proteins and total proteins in rice leaf.Total proteins precipitation with several solvents varied significantly(P<0.05),and the precipitation ability from strong to weak was acetonitrile>acetone>isopropanol>methanol>ethanol.Acetone performed best in the precipitation effect of 16 kinds of target proteins,followed by isopropanol and acetonitrile,and methanol and ethanol represented the worst precipitants.The relative standard deviations of the method were all less than 14%in three orders of magnitude range,with the quantification limit from 0.1 to 2.5 nmol/L,and the contents of 16 kinds of proteins in rice leaf during the grain filling stage ranged from 6.0 to 2818.1μg/g.【Conclusion】SDS could significantly improve the extraction effect of proteins in rice leaf.Acetone and acetonitrile addition generated excellent precipitation effects of proteins,with slight difference in various proteins.Combined with MRM mass spectrometry technology,the absolute quantification method for proteins in rice leaf is featured by wide-linear range,high sensitivity and good repeatability.