Chinese Journal OF Rice Science >
Absolute Quantification of Proteins in Rice Leaf Based on Multiple Reaction Monitoring Mass Spectrometry
#These authors contributed equally to this work
Received date: 2019-05-15
Revised date: 2019-08-01
Online published: 2020-05-10
【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 2818.1 μg/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.
Mingxue CHEN, Huan YANG, Zhaoyun CAO, Youning MA, Zhenzhen CAO, Fangmin CHENG . Absolute Quantification of Proteins in Rice Leaf Based on Multiple Reaction Monitoring Mass Spectrometry[J]. Chinese Journal OF Rice Science, 2020 , 34(3) : 278 -286 . DOI: 10.16819/j.1001-7216.2020.9056
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