生物技术通报 ›› 2020, Vol. 36 ›› Issue (12): 247-255.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0555
收稿日期:
2020-05-09
出版日期:
2020-12-26
发布日期:
2020-12-22
作者简介:
牟永莹,女,硕士研究生,研究方向:蛋白质组学;E-mail:基金资助:
MU Yong-ying(), WANG Dao-ping, CHEN Ming, QIU Li-juan, PAN Ying-hong()
Received:
2020-05-09
Published:
2020-12-26
Online:
2020-12-22
摘要:
构建一种适用于大豆种子蛋白质组样品制备与液相质谱分析的技术体系,为深入研究大豆蛋白组学创造条件。以大豆品种中黄35成熟期种子为材料,分别对常用的蛋白质提取、酶切、液相分离梯度和数据库进行比较分析。结果表明,使用改进的尿素硫脲蛋白提取法配合赖氨酸C端内切酶/胰蛋白酶顺序酶切法制备的质谱分析样品,经90 min纳升级液相梯度分离和质谱分析,最后采用UniProt数据库解谱时,大豆种子蛋白鉴定数目最多。将此方法应用于大豆品种十胜长叶成熟种子样品制备和分析,3次样品制备重复试验共鉴定非冗余蛋白2 244组,其中61%蛋白质可被重复鉴定。该方法具有一定的可靠性和稳定性,适用于大豆种子蛋白质组样品制备和数据分析。
牟永莹, 王道平, 陈明, 邱丽娟, 潘映红. 大豆种子蛋白质组样品制备与数据分析方法[J]. 生物技术通报, 2020, 36(12): 247-255.
MU Yong-ying, WANG Dao-ping, CHEN Ming, QIU Li-juan, PAN Ying-hong. Sample Preparation and Data Analysis Method for Soybean Seed Proteome[J]. Biotechnology Bulletin, 2020, 36(12): 247-255.
液相梯度 | 蛋白 | 肽段 | 二级谱图数 | 肽段-谱图匹配数 |
---|---|---|---|---|
30 min | 709±11 b | 2 890±53 c | 13 015±129 c | 4 747±124 c |
60 min | 1 364 b±4 a | 5 620±22 b | 36 751±173 b | 10 966±30 b |
120 min | 1 396±47 a | 5 771±60 a | 53 129±301 a | 25 256±694 a |
表1 不同液相梯度时间蛋白分离效果比较
液相梯度 | 蛋白 | 肽段 | 二级谱图数 | 肽段-谱图匹配数 |
---|---|---|---|---|
30 min | 709±11 b | 2 890±53 c | 13 015±129 c | 4 747±124 c |
60 min | 1 364 b±4 a | 5 620±22 b | 36 751±173 b | 10 966±30 b |
120 min | 1 396±47 a | 5 771±60 a | 53 129±301 a | 25 256±694 a |
数据库名称 | 数据库总蛋白条目数 | 鉴定蛋白数 | ||
---|---|---|---|---|
有功能注释 | 无功能注释 | |||
Phytozome:Gmax_275_Wm82.a2.v1 | 88 647 | 1 411 | 560 | |
UniProt:Glycine max_cv.Williams 82 | 88 687 | 1 237 | 753 | |
NCBI:Glycine_max_v2.1 | 88 647 | 1 501 | 457 |
表2 不同数据库搜库结果比较
数据库名称 | 数据库总蛋白条目数 | 鉴定蛋白数 | ||
---|---|---|---|---|
有功能注释 | 无功能注释 | |||
Phytozome:Gmax_275_Wm82.a2.v1 | 88 647 | 1 411 | 560 | |
UniProt:Glycine max_cv.Williams 82 | 88 687 | 1 237 | 753 | |
NCBI:Glycine_max_v2.1 | 88 647 | 1 501 | 457 |
[1] |
Jiao C, Gu Z. Itraq-based proteomic analysis reveals changes in response to sodium nitroprusside treatment in soybean sprouts[J]. Food Chemistry, 2019,292:372-376.
URL pmid: 31054689 |
[2] |
Pi EX, Qu LQ, Hu JW, et al. Mechanisms of soybean roots’ tolerances to salinity revealed by proteomic and phosphoproteomic comparisons between two cultivars[J]. Molecular & Cellular Proteomics, 2016,15(1):266-288.
doi: 10.1074/mcp.M115.051961 URL pmid: 26407991 |
[3] |
Sharma M, Gupta SK, Majumder B, et al. Proteomics unravel the regulating role of salicylic acid in soybean under yield limiting drought stress[J]. Plant Physiology and Biochemistry, 2018,130:529-541.
URL pmid: 30098585 |
[4] | 牛宁, 李占军, 金素娟, 等. 大豆蛋白质组学研究进展[J]. 中国油料作物学报, 2014,36(5):667-675. |
Niu N, Li ZJ, Jin SJ, et al. Advances in soybean proteomics[J] Chinese Journal of Oil Crop Sciences, 2014,36(5):667-675. | |
[5] |
Barbosa HS, Arruda SCC, Azevedo RA, et al. New insights on proteomics of transgenic soybean seeds:evaluation of differential expressions of enzymes and proteins[J]. Analytical and Bioanalytical Chemistry, 2012,402(1):299-314.
doi: 10.1007/s00216-011-5409-1 URL |
[6] |
Krishnan HB, Neson RL. Proteomic analysis of high protein soybean(Glycine max)accessions demonstrates the contribution of novel glycinin subunits[J]. Journal of Agricultural and Food Chemistry, 2011,59(6):2432-2439.
doi: 10.1021/jf104330n URL |
[7] | 林杨杰, 赵明, 杨生超, 等. 大豆种子蛋白质组样品制备方法研究[J]. 大豆科学, 2016,35(5):810-817. |
Lin YJ, Zhao M, Yang SC, et al. A Study on Sample Preparations for Proteomics of Soybean Seeds[J]. Soybean Science, 2016,35(5):810-817. | |
[8] |
Park ZY, Russell DH. Thermal denaturation:a useful technique in peptide mass mapping[J]. Analytical Chemistry, 2000,72(11):2667-2670.
URL pmid: 10857653 |
[9] | 林勇. 蛋白质组学新方法的建立及其在膜蛋白质组研究中的应用[D]. 长沙:湖南师范大学, 2010. |
Lin Y. Development of new proteomics methods and their applications to the analysis of membrane proteome[D]. Changsha:Hunan Normal University, 2010. | |
[10] | 李倩, 冯钰, 谭敏佳, 等. 赖氨酸c端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估[J]. 分析化学, 2017,45(3):316-321. |
Li Q, Feng Y, Tan MJ, et al. Evaluation of endoproteinase Lys-C/Trypsin sequential digestion used in proteomics sample preparation[J]. Chinese Journal of Analytical Chemistry, 2017,45(3):316-321. | |
[11] |
Wang H, Yang YL, Li YX, et al. Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome[J]. J Proteome Res, 2015,14(2):829-838.
doi: 10.1021/pr500882h URL pmid: 25455107 |
[12] |
Woehlbrand L, Rabus R, Blasius B, et al. Influence of nanolc column and gradient length as well as ms/ms frequency and sample complexity on shotgun protein identification of marine bacteria[J]. Journal of Molecular Microbiology and Biotechnology, 2017,27(3):199-212.
URL pmid: 28850952 |
[13] | Goodstein DM, Shu S, Howson R, et al. Phytozome:a comparative platform for green plant genomics[J]. Nucleic Acids Research, 2012,40(d1):d1178-d1186. |
[14] | 罗静初. uniprot蛋白质数据库简介[J]. 生物信息学, 2019,17(3):131-144. |
Luo JC. A brief introduction to UniProt[J]. Chinese Journal of Bioinformatics, 2019,17(3):131-144. | |
[15] | 饶冬梅. NCBI数据库及其资源的获取[J]. 科技视界, 2013(7):53-54. |
Rao DM. Analysis of resources access on NCBI database[J]. Science & Technology Vision, 2013(7):53-54. | |
[16] |
Natarajan S, Xu CP, Caperna TJ, et al. Comparison of protein solubilization methods suitable for proteomic analysis of soybean seed proteins[J]. Anal Biochem, 2005,342(2):214-220.
URL pmid: 15953580 |
[17] |
Wisniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation method for proteome analysis[J]. Nature Methods, 2009,6(5):359-360.
URL pmid: 19377485 |
[18] |
Cox J, Neuhauser N, Michalski A, et al. Andromeda:a peptide search engine integrated into the maxquant environment[J]. Journal of Proteome Research, 2011,10(4):1794-1805.
URL pmid: 21254760 |
[19] |
Min CW, Gupta R, Agrawal GK, et al. Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach[J]. Expert Rev Proteomics, 2019,16(9):795-804.
URL pmid: 31398080 |
[20] |
Oskuei BK, Yin X, Hashiguchi A, et al. Proteomic analysis of soybean seedling leaf under waterlogging stress in a time-dependent manner[J]. Biochimica et Biophysica Acta-Proteins and Proteomics, 2017,1865(9):1167-1177.
doi: 10.1016/j.bbapap.2017.06.022 URL pmid: 28666670 |
[21] |
Wang X, Khodadadi E, Fakheri B, et al. Organ-specific proteomics of soybean seedlings under flooding and drought stresses[J]. Journal of Proteomics, 2017,162:62-72.
doi: 10.1016/j.jprot.2017.04.012 URL pmid: 28435105 |
[22] |
Wiśniewski JR. Filter-aided sample preparation:the versatile and efficient method for proteomic analysis[J]. Methods Enzymol, 2017,585:15-17.
doi: 10.1016/bs.mie.2016.09.013 URL pmid: 28109427 |
[23] | Jez E, Lestan D. Edta retention and emissions from remediated soil[J]. Chemosphere, 2016,151:202-209. |
[24] | Betancourt LH, Sanchez A, Pla I, et al. Quantitative assessment of urea in-solution lys-c/trypsin digestions reveals superior performance at room temperature over traditional proteolysis at 37 degrees c[J]. Journal of Proteome Research, 2018,17(7):2556-2561. |
[25] | Hakobyan A, Schneider MB, Liesack W, et al. Efficient tandem lysc/trypsin digestion in detergent conditions[J]. Proteomics, 2019,19(20):6. |
[26] |
Erde J, Loo RRO, Loo JA. Enhanced FASP(eFASP)to increase proteome coverage and sample recovery for quantitative proteomic experiments[J]. Journal of Proteome Research, 2014,13(4):1885-1895.
URL pmid: 24552128 |
[27] |
Ni MW, Wang L, Chen W, et al. Modified filter-aided sample preparation(FASP)method increases peptide and protein identifications for shotgun proteomics[J]. Rapid Communications in Mass Spectrometry, 2017,31(2):171-178.
URL pmid: 27794190 |
[28] |
Loraine J, Alhumaidan O, Bottrill AR, et al. Efficient protein digestion at elevated temperature in the presence of sodium dodecyl sulfate and calcium ions for membrane proteomics[J]. Analytical Chemistry, 2019,91(15):9516-9521.
URL pmid: 31259536 |
[29] |
Hsieh EJ, Bereman MS, Durand S, et al. Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples[J]. Journal of the American Society for Mass Spectrometry, 2013,24(1):148-153.
URL pmid: 23197307 |
[30] |
Baba M, Ohyama K, Kishikawa N, et al. Optimization of separation and digestion conditions in immune complexome analysis[J]. Analytical Biochemistry, 2013,443(2):181-186.
doi: 10.1016/j.ab.2013.08.026 URL pmid: 24012793 |
[31] |
Xu P, Duong DM, Peng J. Systematical optimization of reverse-phase chromatography for shotgun proteomics[J]. Journal of Proteome Research, 2009,8(8):3944-3950.
doi: 10.1021/pr900251d URL pmid: 19566079 |
[32] |
Xu XP, Liu H, Tian L, et al. Integrated and comparative proteomics of high-oil and high-protein soybean seeds[J]. Food Chemistry, 2015,172:105-116.
URL pmid: 25442530 |
[1] | 赵明明, 唐殷, 郭磊周, 韩佳慧, 葛佳茗, 孟勇, 平淑珍, 周正富, 王劲. Lon1蛋白酶参与耐辐射异常球菌高温胁迫及细胞分裂的功能研究[J]. 生物技术通报, 2022, 38(5): 149-158. |
[2] | 李兵娟, 郑璐, 沈仁芳, 兰平. 拟南芥RPP1A参与幼苗生长的蛋白质组学分析[J]. 生物技术通报, 2022, 38(2): 10-20. |
[3] | 王智博, 王道平, 苗兰, 李瑛, 潘映红, 刘建勋. 血液样本蛋白质组分析方法的比较研究[J]. 生物技术通报, 2021, 37(8): 307-318. |
[4] | 刘静, 李亚超, 周梦岩, 吴鹏飞, 马祥庆, 李明. 植物蛋白质翻译后修饰组学研究进展[J]. 生物技术通报, 2021, 37(1): 67-76. |
[5] | 郑璐, 沈仁芳, 兰平. 植物非组蛋白赖氨酸乙酰化修饰的蛋白质组学研究进展[J]. 生物技术通报, 2021, 37(1): 77-89. |
[6] | 孟丽娜, 彭春莹, 李铁栋, 李博生. 基于蛋白质组学对螺旋藻砷胁迫响应机制的研究[J]. 生物技术通报, 2020, 36(4): 107-116. |
[7] | 李堃, 刘悦, 黄鹏, 杨智昉, 胡茜, 张颖, 李志宏, 吕叶辉, 梁乐. 小鼠精原细胞分化的蛋白质组学研究[J]. 生物技术通报, 2020, 36(3): 168-176. |
[8] | 张良, 陈小青, 宋佳宇, 毛然然, 姜倩雯, 林向民. 巴洛沙星胁迫下大肠杆菌的比较蛋白质组学研究[J]. 生物技术通报, 2019, 35(3): 103-109. |
[9] | 兰玉婷, 王双蕾, 李征珍, 冯金朝, 王晓东, 石莎. 沙冬青属植物响应非生物胁迫的蛋白质组学研究进展[J]. 生物技术通报, 2019, 35(1): 112-119. |
[10] | 谭君, 牟云, 周光普, 周永顺, 徐杰, 高剑峰. 水胁迫下沙漠小球藻蛋白质组学分析[J]. 生物技术通报, 2018, 34(10): 207-216. |
[11] | 牟永莹,顾培明,马博,闫文秀,王道平,潘映红. 基于质谱的定量蛋白质组学技术发展现状[J]. 生物技术通报, 2017, 33(9): 73-84. |
[12] | 邵贵芳, 张凡, 王姣, 赵凯, 莫云容, 邓明华. 辣椒雄性不育的研究进展[J]. 生物技术通报, 2017, 33(8): 7-13. |
[13] | 付晨熙, 肖自华, 高飞, 周宜君. 干旱胁迫下蒙古沙冬青叶片蛋白质组学研究[J]. 生物技术通报, 2017, 33(6): 69-80. |
[14] | 余乐正, 柳凤娟, 吴正雨, 冉小强. 分泌蛋白质组学在肿瘤标志物中的研究进展[J]. 生物技术通报, 2017, 33(3): 12-21. |
[15] | 宋雁超, 安飞飞, 薛晶晶, 秦于玲, 李开绵, 陈松笔. 木薯栽培种ZM-Seaside和花叶变种块根蛋白组学分析[J]. 生物技术通报, 2017, 33(3): 78-85. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||