[1]甄 艳,郑秀化,施季森,等.适合杨树叶片蛋白质组学样品制备方法的比较[J].南京林业大学学报(自然科学版),2018,42(02):033-39.[doi:10.3969/j.issn.1000-2006.201706063]
 ZHEN Yan,ZHENG Xiuhua,SHI Jisen*.Comparison of sample preparation methods suitable for poplar leaf proteomics[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(02):033-39.[doi:10.3969/j.issn.1000-2006.201706063]
点击复制

适合杨树叶片蛋白质组学样品制备方法的比较
分享到:

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
42
期数:
2018年02期
页码:
033-39
栏目:
研究论文
出版日期:
2018-03-20

文章信息/Info

Title:
Comparison of sample preparation methods suitable for poplar leaf proteomics
文章编号:
1000-2006(2018)02-0033-07
作者:
甄 艳郑秀化施季森
南方现代林业协同创新中心,林木遗传与生物技术省部共建教育部重点实验室,南京林业大学林学院,江苏 南京 210037
Author(s):
ZHEN Yan ZHENG Xiuhua SHI Jisen*
Co-innovation Center for the Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology of the Ministry of Education, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
关键词:
核酮糖-15-二磷酸羧化-加氧酶 聚乙二醇分级 杨树叶片 双向电泳
Keywords:
Keywords:Rubisco PEG fractionation poplar leaves two-dimensional electrophoresis(2-DE)
分类号:
S792.11
DOI:
10.3969/j.issn.1000-2006.201706063
文献标志码:
A
摘要:
【目的】为了提高植物叶片中低丰度蛋白质的检测效率,建立有效的杨树叶片蛋白质组学分析的蛋白质提取方法。【方法】利用三氯乙酸-丙酮结合PEG分级提取方法和SDS-酚结合PEG分级提取方法对杨树叶片蛋白质进行提取,并采用SDS-PAGE和双向凝胶电泳(2-DE)对提取结果进行评价。【结果】SDS-PAGE实验结果显示,三氯乙酸-丙酮结合PEG方法优于SDS-酚结合PEG分级方法。三氯乙酸-丙酮结合PEG分级和非结合PEG分级提取蛋白质的双向电泳结果表明,PEG分级能有效地去除高丰度蛋白质Rubisco,提高低丰度蛋白质的检测。三氯乙酸-丙酮结合PEG分级法检测的蛋白质比非结合PEG分级法多90个蛋白质点。【结论】以LTQ-Orbitrap XL分析三氯乙酸-丙酮结合PEG分级分离的一些蛋白质显示,三氯乙酸-丙酮结合PEG分级对于2-DE和MS鉴定低丰度蛋白质是一种有效的分离方法。
Abstract:
【Objective】In order to improve the detection of low abundance proteins in plant leaves, the effective extraction methods were established in poplar leaves for proteomics analysis.【Method】 Trichloroacetic acid(TCA)-acetone coupled with polyethylene glycol(PEG)fractionation and sodium dodecyl sulfate(SDS)-phenol coupled with PEG fractionation were evaluated, combined with SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and two-dimensional electrophoresis(2-DE).【Result】The SDS-PAGE results showed that TCA-acetone coupled with PEG fractionation is superior to SDS-phenol coupled with the PEG fractionation method. Further, proteins with PEG fractionation and no PEG fractionation coupled with TCA-acetone were compared by using 2-DE, and the gel maps displayed that PEG fractionation is a good way to deplete the Rubiscos and improve the detection of low abundant proteins. The PEG fractionation method can detect nearly 90 more spots than nonfractionation can. 【Conclusion】LTQ-Orbitrap XL was used to identify several proteins from the PEG fractionation coupled with TCA-acetone. The TCA-acetone coupled with PEG fractionation is an effective approach for the detection of 2-DE analysis and mass spectroscopy(MS)identification of lower abundance proteins.

参考文献/References:

[1] CELLAR N A, KUPPANNAN K, LANGHORST M L, et al. Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase-oxygenase [J]. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2008, 861(1):29-39. DOI:10.1016-j.jchromb.2007.11.024.
[2] GIAVALISCO P, NORDHOFF E, KREITLER T, et al. Proteome analysis of Arabidopsis thaliana by two-dimensional gel electrophoresis and matrix-assisted laser desorption-ionisation-time of flight mass spectrometry [J]. Proteomics, 2005, 5(7):1902-1913. DOI:10.1002-pmic.200401062.
[3] LEE D G, AHSAN N, LEE S H, et al. An approach to identify cold-induced low-abundant proteins in rice leaf [J]. Comptes Rendus Biologies, 2007, 330(3):215-225.
[4] WIDJAJA I, NAUMANN K, ROTH U, et al. Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense [J]. Proteomics, 2009, 9(1):138-147. DOI:10.1016-j.crvi.2007.01.001.
[5] ATHA D H, INGHAM K C. Mechanism of precipitation of proteins by polyethylene glycols: analysis in terms of excluded volume [J]. The Journal of Biological Chemistry, 1981, 256(23):12108-12117. PMID:7298647
[6] JUCKES I R. Fractionation of proteins and viruses with polyethylene glycol[J]. Biochimica et Biophysica Acta, 1971, 229(3):535-546. PMID:5103022
[7] XI J, WANG X, LI S, et al. Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome [J]. Phytochemistry, 2006, 67(21):2341-2348. DOI:10.1016-j.phytochem.2006.08.005.
[8] AHSAN N, LEE D G, LEE S H, et al. A comparative proteomic analysis of tomato leaves in response to waterlogging stress [J]. Physiologia Plantarum, 2007, 131(4):555-570. DOI:10.1111-j.1399-3054.2007.00980.x.
[9] FALVO S, ACQUADRO A, ALBO A G, et al. Proteomic analysis of PEG fractionated UV-C stress-response proteins in globe Artichoke[J]. Plant Molecular Biology Reporter, 2012, 30(1):111-122. DOI.org-10.1007-s11105-011-0325-2
[10] KIM S T, CHO K S, JANG Y S, et al. Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays [J]. Electrophoresis, 2001, 22(10):2103-2109. DOI:10.1002-1522-2683(200106)22:10<2103::AID-ELPS2103>3.0.CO; 2-W.
[11] ZHANG A, LU Q, YIN Y, et al. Comparative proteomic analysis provides new insights into the regulation of carbon metabolism during leaf senescence of rice grown under field conditions[J]. Journal of Plant Physiology, 2010, 167(16):1380-1389. DOI:10.1016-j.jplph.2010.05.011
[12] SHEN J W, BUKO A. Rapid identification of proteins in polyethylene glycol-containing samples using capillary electrophoresis electrospray mass spectrometry[J]. Analytical Biochemistry, 2002, 311(1):80-83. PMID:12441156
[13] HESSE A M, MARCELO P, ROSSIER J, et al. Simple and universal tool to remove on-line impurities in mono-or two-dimensional liquid chromatography-mass spectrometry analysis [J]. Journal of Chromatography A, 2008, 1189(1/2):175-182. DOI:10.1016-j.chroma.2007.12.060.
[14] ARYAL U K, KROCHKO J E, ROSS A R S. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry [J]. Journal of Proteome Research, 2012, 11(1):425-437. DOI:10.1021-pr200917t.
[15] ZHEN Y, QI J L, WANG S S, et al. Comparative proteome analysis of differentially expressed proteins induced by Al toxicity in soybean [J]. Physiologia Plantarum, 2007, 131(4):542-554. DOI:10.1111-j.1399-3054.2007.00979.x.
[16] UEMATSU K, SUZUKI N, IWAMAE T, et al. Increased fructose 1,6-bisphosphate aldolase in plastids enhances growth and photosynthesis of tobacco plants [J]. Journal of Experimental Botany, 2012, 63(8):3001-3009. DOI:10.1093-jxb-ers004.
[17]PARK J I, ISHIMIZU T, SUWABE K, et al. UDP-Glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana [J]. Plant and Cell Physiology, 2010, 51(6):981-996. DOI:10.1093-pcp-pcq057.
[18] CHIVASA S, TOME D F A, SLABAS A R. UDP-Glucose pyrophosphorylase is a novel plant cell death regulator [J]. Journal of Proteome Research, 2013, 12(4):1743-1753. DOI:10.1021-pr3010887.

备注/Memo

备注/Memo:
基金项目:国家高技术研究发展计划(2013AA102705); 江苏高校优势学科建设工程资助项目(PAPD) 第一作者:甄艳(zhenyongni30@aliyun.com),副教授,博士。*通信作者:施季森(jisen@njfu.edu.cn),教授。
更新日期/Last Update: 2018-06-12