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

doi:10.16819/j.1001-7216.2020.9056

Abstract

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

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

CLC Number:

S511.01

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.

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.

Figures/Tables 6

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.

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

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%.

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%.

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%.

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

References 28

[1]	钱小红. 定量蛋白质组学分析方法[J]. 色谱, 2013, 31(8): 719-723.
	Qian X H.Quantitative proteomics analysis[J]. Chinese Journal of Chromatography, 2013, 31(8): 719-723. (in Chinese with English abstract)
[2]	齐林玉, 姜淼, 郭艳, 王迎, 陈平. 质谱MRM技术结合稳定同位素标记法的应用进展[J]. 生命科学研究, 2015, 19(5): 444-451.
	Qi L Y, Jiang M, Guo Y, Wang Y, Chen P.Progresses on application of MS MRM technology combining with stable isotope labeling[J]. Life Science Research, 2015, 19(5): 444-451. (in Chinese with English abstract)
[3]	Veronika V, Zdenek S.A review on mass spectrometry-based quantitative proteomics: Targeted and data independent acquisition[J]. Analytica Chimica Acta, 2017, 964: 7-23.
[4]	王素兰, 高华萍, 张菁, 叶翔. 基于稳定同位素标记和平行反应监测的蛋白质组学定量技术用于肝癌生物标志物的筛选和验证[J]. 色谱, 2017, 35(9): 934-940.
	Wang S L, Gao H P, Zhang J, Ye X.Stable isotope labeling and parallel reaction monitoring-based proteomic quantification for biomarker screening and validation of hepatocellular carcinoma[J]. Chinese Journal of Chromatography, 2017, 35(9): 934-940. (in Chinese with English abstract)
[5]	Chen Y T, Chen H W, Wu C F, Chu L J, Chiang W F, Wu C C, Yu J S, Tsai C H, Liang K H, Chang Y S, Wu M, Ou Yang W T. Development of a multiplexed liquid chromatography multiple-reaction-monitoring mass spectrometry (LC-MRM/MS) method for evaluation of salivary proteins as oral cancer biomarkers[J]. Molecular Cellular Proteomics, 2017, 16(5): 799-811.
[6]	Zhang J, Lai S, Cai Z, Chen Q, Huang B, Ren Y.Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard[J]. Analytica Chimica Acta, 2014, 4(829): 33-39.
[7]	Planque M, Arnould T, Delahaut P, Renardb P, Dieub M, Gillard N.Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory[J]. Food Chemistry, 2019, 274: 35-45.
[8]	Rautengarten C, Ebert B, Heazlewood J L.Absolute quantitation of in vitro expressed plant membrane proteins by targeted proteomics (MRM) for the determination of kinetic parameters[J]. Methods in Molecular Biology, 2018, 1696: 217-234.
[9]	Gong C, Zheng N, Zeng J, Aubry A F, Arnold M E.Post-pellet-digestion precipitation and solid phase extraction: A practical and efficient workflow to extract surrogate peptides for ultra-high performance liquid chromatography-tandem mass spectrometry bioanalysis of a therapeutic antibody in the low ng/mL range[J]. Journal of Chromatography A, 2015, 1424: 27-36.
[10]	Wu X, Xiong E, Wang W, Scali M, Cresti M.Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis[J]. Nature Protocols, 2014, 9(2): 362-374.
[11]	Luís I M, Alexandre B M, Oliveira M M, Abreu I A.Selection of an appropriate protein extraction method to study the phosphoproteome of maize photosynthetic tissue[J/OL].PLoS One, 2016, 11(10): e0164387.
[12]	Kilambi H V, Manda K, Sanivarapu H, Maurya V K, Sharma R, Sreelakshmi Y.Shotgun proteomics of tomato fruits: Evaluation, optimization and validation of sample preparation methods and mass spectrometric parameters [J/OL]. Frontiers in Plant Science, 2016, 7: 969.
[13]	Bereman M S, Egertson J D, MacCoss M J. Comparison between procedures using SDS for shotgun proteomic analyses of complex samples[J]. Proteomics, 2011, 11(14): 2931-5.
[14]	Yang L, Ji J, Harris-Shultz K R, Wang H, Wang H, Abd-Allah E F, Luo Y, Hu X Y. The dynamic changes of the plasma membrane proteins and the protective roles of nitric oxide in rice subjected to heavy metal cadmium stress[J/OL].Frontiers in Plant Science, 2016, 7: 190.
[15]	高欢欢, 马有宁, 林晓燕, 柴爽爽, 秦美玲, 张涵彤, 何巧, 陈铭学. 亲水作用-反相二维液相色谱串联质谱法鉴定水稻蛋白质[J]. 分析化学, 2018, 46(5): 650-657.
	Gao H H, Ma Y N, Lin X Y, Chai S S, Qin M L, Zhang H T, He Q, ChenM X. Development of two-dimensional liquid chromatography coupled with tandem mass spectrometry for identification of extracted proteins of rice leaves: Hydrophilic interaction-reversed-phase approach[J]. Chinese Journal of Analytical Chemistry, 2018, 46(5): 650-657. (in Chinese with English abstract)
[16]	Ippoushi K, Sasanuma M, Oike H, Kobori M, Maeda-Yamamoto M.Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard[J]. Food Chemistry, 2015, 173: 238-242.
[17]	Wisniewski J R, Zouqman A, Nagaraj N, Mann M.Universal sample preparation method for proteome analysis[J]. Nature Methods, 2009, 6(5): 359-362.
[18]	Bru-Martínez R, Martínez-Márquez A, Morante-Carriel J, Sellés-Marchart S, Martínez-Esteso M J, Pineda-Lucas J L, Luque I. Targeted quantification of isoforms of a thylakoid-bound protein: MRM method development[J]. Methods in Molecular Biology, 2018, 1696: 147-162.
[19]	An B, Zhang M, Johnson R W, Qu J.Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues[J]. Analytical Chemistry, 2015, 87(7): 4023-4029.
[20]	Santa C, Anjo S I, Manadas B.Protein precipitation of diluted samples in SDS-containing buffer with acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone approach[J]. Proteomics, 2016, 16(13): 1847-1851.
[21]	Botelho D, Wall M J, Vieira D B, Fitzsimmons S, Liu F, Doucette A.Top-down and bottom-up proteomics of SDS containing solutions following mass-based separation[J]. Journal of Proteome Research, 2010, 9(6): 2863-2870.
[22]	Zhang N, Chen R, Young N, Wishart D, Winter P, Weiner J H, Li L.Comparison of SDS and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2D LC-MS/MS[J]. Proteomics, 2007, 7(4): 484-493.
[23]	Ilavenil S, Al-Dhabi N A, Srigopalram S, Kim Y O, Agastian P, BaaruKi R, Choon Choi K C, Arasu M V, Park C G, Park K H. Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry[J]. Proteome Science, 2016, 14(1): 11.
[24]	Ouyang Z, Furlong M T, Wu S, Sleczka B, Tamura J, Wang H, Suchard S, Suri A, Olah T, Tymiak A, Jemal M.Pellet digestion: A simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteinsin plasma[J]. Bioanalysis, 2012, 4(1): 17-28.
[25]	Srivastava A K. A Comparison of protein extraction methods using organic solvents for secretome of aspergillus fumigatus strain (MTCC 1811). International Journal of Scientific Research, 2015, 4(6): 2277-8179.
[26]	张冬梅, 马慧萍, 贾正平. 纳升级反相液相色谱串联质谱法分析海马蛋白质组[J]. 药物分析杂志, 2018, 38(1): 118-123.
	Zhang D M, Ma H P, Jia Z P.Proteomic analysis of Hippocampus using nanoflow reversed phase liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Pharmaceutical Analysis, 2018, 38(1): 118-123. (in Chinese with English abstract)
[27]	Rezeli M, Sjödin K, Lindberg H, Gidlöf O, Lindahl B, Jernberg T, Spaak J, Erlinge D, Marko-Varga G.Quantitation of 87 proteins by nLC-MRM/MS in human plasma: workflow for large-scale analysis of biobank samples[J]. Journal of Proteome Research, 2017, 16(19): 3242-3254.
[28]	Hoofnagle A N, Becker J O, Oda M N, Cavigiolio G, Mayer P, Vaisar T.Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixture[J]. Clinical Chemistry, 2012, 58(4): 777-781.