基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法

doi:10.16819/j.1001-7216.2020.9056

中国水稻科学 ›› 2020, Vol. 34 ›› Issue (3): 278-286.DOI: 10.16819/j.1001-7216.2020.9056

• 实验技术 • 上一篇

基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法

陈铭学^1,²^,^#, 杨欢²^,^#, 曹赵云², 马有宁², 曹珍珍², 程方民^1,^*()

¹浙江大学农业与生物技术学院,杭州 310058
²中国水稻研究所,杭州 310006

收稿日期:2019-05-15 修回日期:2019-08-01 出版日期:2020-05-15 发布日期:2020-05-10
通讯作者: 程方民
作者简介:
^#共同第一作者
基金资助:
国家自然科学基金资助项目（31701408）;国家水稻产业体系资助项目（CARS-01-47）;中国农业科学院农业科技创新工程资助项目（CAAS- ZDRW202011）;中央级公益性科研院所基本科研业务费专项（Y2019PT19-01）

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

Mingxue CHEN^1,²^,^#, Huan YANG²^,^#, Zhaoyun CAO², Youning MA², Zhenzhen CAO², Fangmin CHENG^1,^*()

¹College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
²China National Rice Research Institute, Hangzhou 310006, China

Received:2019-05-15 Revised:2019-08-01 Online:2020-05-15 Published:2020-05-10
Contact: Fangmin CHENG
About author:
^#These authors contributed equally to this work

摘要/Abstract

摘要：

【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6.0~2818.1 μg/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。

关键词: 水稻叶片, 蛋白质, 多反应监测, SOD法, 绝对定量

Abstract:

【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.

Key words: rice leaf, protein, multiple reaction monitoring, SOD method, absolute quantification

中图分类号:

S511.01

陈铭学, 杨欢, 曹赵云, 马有宁, 曹珍珍, 程方民. 基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法[J]. 中国水稻科学, 2020, 34(3): 278-286.

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.

图/表 6

图1 16种特征肽段的总离子流

Fig. 1. Total ion chromatogram of 16 kinds of signature peptides. 1, P12085; 2, Q40677; 3, P0C2Z6; 4, Q8H8D6; 5, Q8GTK4; 6, A3BLC3; 7, Q650W6; 8, Q9ZP20; 9, Q0D5P8; 10, Q948T6; 11, Q8S718; 12, Q7X8A1; 13, Q0DEU8; 14, Q7XV11; 15, Q7XZW5; 16, Q69S39.

表1 16种特征肽段的线性方程、相关系数及仪器检出限与定量限

Table 1 Linear equations, correlation coeffcients(r), limits of detection(LOD) and limits of quantification (LOQ) of the 16 kinds of signature peptides.

蛋白质序列号 Accession	线性方程 Linear equation	相关系数 r	线性范围 Linear range/(nmol·L^-1)	检出限 LOD/(nmol·L^-1)	定量限 LOQ/(nmol·L^-1)
Q40677	y=1.97e⁴ x + 5.21e⁵	0.9965	0.5~250.0	0.17	0.5
P12085	y=1.75e⁴ x + 3.11e⁵	0.9990	0.6~300.0	0.20	0.6
A3BLC3	y=0.98e⁴ x + 5.47e⁵	0.9974	0.3~200.0	0.10	0.3
Q9ZP20	y=3.25e³ x + 2.94e⁵	0.9983	1.0~1000.0	0.33	1.0
Q0D5P8	y=4.14e³ x + 6.54e⁵	0.9909	2.5~1000.0	0.80	2.5
Q7X8A1	y=8.59e⁴ x + 2.95e⁶	0.9993	0.5~250.0	0.17	0.5
Q0DEU8	y=2.14e³ x + 4.87e⁵	0.9974	0.5~250.0	0.17	0.5
Q7XV11	y=4.59e³ x + 2.65e⁵	0.9986	0.1~100.0	0.03	0.1
Q7XZW5	y=2.56e³ x + 2.46e⁵	0.9959	0.5~250.0	0.17	0.5
P0C2Z6	y=9.13e³ x + 4.48e⁴	0.9967	0.7~300.0	0.23	0.7
Q650W6	y=7.54e³ x + 6.21e⁵	0.9987	0.4~200.0	0.13	0.4
Q69S39	y=1.58e³ x + 4.64e⁵	0.9988	1.7~1000.0	0.56	1.7
Q8GTK4	y=2.51e⁴ x + 3.36e⁶	0.9989	1.8~1000.0	0.60	1.8
Q8H8D6	y=5.57e⁴ x + 3.78e⁵	0.9991	0.4~200.0	0.13	0.4
Q8S718	y=3.45e⁴ x + 2.49e⁵	0.9988	0.3~200.0	0.10	0.3
Q948T6	y=4.44e³ x + 4.25e⁵	0.9982	0.4~200.0	0.13	0.4

图2 提取缓冲液中不同浓度SDS对16种水稻叶片蛋白提取效果影响图中数据为均值±标准差（n=3）;不同字母表示在0.05水平上有显著性差异,而具相同字母表示无显著性差异。纵坐标为蛋白质相对提取百分比,以未加SDS的提取液对应的各目标蛋白提取量为100%计算。

Fig. 2. Influence of different concentrations of SDS in extraction buffer on the extraction effects of 16 kinds of proteins in rice leaves. The data in the figure are mean ± SD (n=3), and different letters indicate significant differences at P<0.05, while the same letters mean no significant difference. The ordinate is the relative extraction percentage of protein with the SDS-free extraction amount of each target protein as 100%.

图3 五种有机试剂对16种水稻叶片蛋白沉淀效果比较图中数据为均值±标准差（n=3）,不同字母表示在0.05水平上有显著性差异,而具相同字母表示无显著性差异。纵坐标为蛋白质相对提取百分比,以乙腈沉淀对应的各目标蛋白提取量为100%计。

Fig. 3. Comparison of precipitation effects of 16 kinds of proteins in rice leaves among five different organic reagents. The data in the figure are mean ± SD (n=3), and different letters indicate significant differences at P<0.05, while the same letters mean no significant difference. The ordinate is the relative extraction percentage of target protein using various precipitation reagents, with acetonitrile-precipitated protein amount as 100%.

图4 SOD法、酚法和TCA-丙酮沉淀法对16种水稻叶片蛋白的提取效果比较图中数据为均值±标准差（n=3）,不同字母表示在0.05水平上有显著性差异,而具相同字母表示无显著性差异。纵坐标为蛋白质相对提取量,以酚法对应的各目标蛋白提取量为100%计。

Fig. 4. Comparison of extraction effects of 16 proteins in rice leaves by SOD, phenol and TCA-acetone precipitation methods. The data in the figure are as mean ± SD (n=3), and different letters indicate significant differences at P<0.05, while the same letters mean no significant difference. The ordinate is the relative extraction percentage of target protein by various methods, with protein amount extracted by phenol method as 100%.

表2 SOD法测定水稻16种叶片蛋白质绝对含量(n=6)

Table 2 Determination of absolute contents of 16 kinds of proteins in rice leaves by SOD method(n=6).

蛋白序列号Accession	测定含量 Measured content/(μg·g^-1)	相对标准偏差 RSD/%
Q40677	589.5±57.5	9.8
P12085	28.5±2.3	8.2
A3BLC3	43.3±3.9	8.9
Q9ZP20	50.2±3.6	7.1
Q0D5P8	606.9±56.0	9.2
Q7X8A1	328.3±27.1	8.3
Q0DEU8	285.8±30.1	10.5
Q7XV11	20.6±1.7	8.2
Q7XZW5	153.2±14.0	9.1
P0C2Z6	67.9±4.8	7.0
Q650W6	25.5±2.9	11.3
Q69S39	2818.1±219.9	7.8
Q8GTK4	109.6±8.1	7.4
Q8H8D6	6.0±0.4	6.2
Q8S718	11.7±0.9	7.4
Q948T6	11.8±1.7	13.9

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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

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