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研究生: 張郡倫
Chun-Lun Chang
論文名稱: 評估胜肽分餾策略對人類肝癌腫瘤細胞PLC/PRF/5 之蛋白質體分析
Evaluation of Peptide Fractionation Strategies Used in Proteomic Analysis of PLC/PRF/5 Cell Line
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2011
畢業學年度: 100
語文別: 中文
論文頁數: 105
中文關鍵詞: 二維液相層析胜肽分餾策略強陽離子交換樹脂OFFGEL正交性電噴灑串聯式質譜
英文關鍵詞: Two-dimensional liquid chromatography, Peptide fractionation, Strong cation exchange, Solution isoelectric focusing, Orthogonality, ESI-MS/MS
論文種類: 學術論文
相關次數: 點閱:336下載:2
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  • 霰彈槍蛋白質體學(shotgun proteomics)是利用蛋白酶水解後的複雜胜肽樣品搭配串聯式質譜分析,即可準確鑑定複雜樣品中的蛋白質體,因此有效降低樣品複雜度的胜肽分餾方法在蛋白質體學的分析上是一門重要的技術。然而過去的研究中,許多文獻比較不同串聯式二維分析方法效率的文獻,本篇比較質譜分析前的四種胜肽分餾策略包括:強陽離子交換樹脂(SCX)、親水作用層析(HILIC)、鹼性逆相層析(alkaline-RP)、等電點聚焦分級分離儀(OFFGEL)和一控制組酸性逆相層析(acidic-RP),我們預期結合不同分離選擇性方法可達到良好的分離效率。根據分餾法不同的分離正交性(orthogonality)與液相層析效能,樣品去鹽後利用SCX x RP-LC分析得到最佳的蛋白質鑑定數目(佔總體蛋白質鑑定的96.54%),接下來是HILIC x RP-LC和alkaline-RP x RP-LC分析。值得注意的是去鹽後樣品利用SCX x RP-LC分析比樣品未經去鹽鑑定到更多的蛋白質(分別是1,990與1,375)。另外我們也發現結合所有胜肽分餾策略結果,鑑定到未重複胜肽(unique peptide)數目大幅增加,而鑑定的蛋白質數目並無顯著增加,因此對應蛋白質的未重複胜肽數目增加(胜肽數/蛋白質比例: 7.3),進而提升蛋白質身分鑑定的證據。不僅如此,鑑定到專一胜肽數目增加,對於後轉譯修飾的偵測例如: 磷酸化,與植基於胜肽之同位素標記定量方法例如: ICAT, iTRAQ, SILAC和TMT等,都有相當大的益處。此結果增加單一胜肽數對應蛋白質不僅提供良好的定量與定性資訊,也增加蛋白質序列的覆蓋率,可得到更高的可信度。

    Shotgun approachhas been a commonlyapplied method for proteome analysis. In order to reduce the samplecomplexity prior to tandem mass spectrometry,peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few in which the relative separation efficiencies of 2D methodologies using complex biological samples are compared, a systematic evaluation was conducted in this study. Four different fractionation methods, including SCX(strongcation exchange), HILIC(hydrophilic interaction liquid chromatography), alkaline-RP and solution IEF prior to LC-MS/MS analysis wereinvestigated using PLC/PRF/5 cell lysate. Based on theircomplementary selectivities, it isexpected they canprovidea more comprehensive separation.SCX x RP-LC, using desalted samples, permitted the greatest number of proteins to be identifiedin this study(96.54% of the total proteins were identified). The result was followed closely by HILIC x RP-LC and alkaline-RP x RP-LC. It is noteworthy that, when SCX x RP-LC was used after desalting the sample, significantly more proteins were able to be identified, compared with the non-desalted sample(1990 and 1375, respectively). It isalso found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that were identified, compared with only a small increase in protein levels(average unique peptides/protein ratio is 7.3). The increased number of distinct peptides that can be identified is especially beneficial, not only
    for unequivocally identifying proteins but also for proteomic studies involving post-translational modifications and peptide-based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.

    謝誌 I 目錄 II 圖目錄 V 表目錄 VII 中文摘要 VIII Abstract XI 縮寫 IX 第一章 緒論 1 一、前言 1 二、液相色譜法 4 三、常見層析方法介紹 5 1.逆相層析法 5 2.親水作用層析法 6 3.強陽離子交換層析法 6 四、二維液相層析法 6 五、等電點聚焦電泳 9 六、高效能液相層析搭配質譜的發展 9 七、應用質譜鑑定蛋白質序列 11 八、研究動機 13 第二章 實驗材料與方法 14 一、樣品 14 二、藥品與試劑 14 三、實驗材料 15 四、儀器設備 16 五、樣品純化濃縮 17 六、蛋白質濃度測定 17 七、蛋白質水解 18 八、胜肽樣品去鹽純化 18 九、第一維分餾策略 19 9-1.酸性逆相層析法 19 9-2-1.鹼性逆相層析法 20 9-2-2.鹼性逆相層析法 20 9-3.親水作用層析法 21 9-4.強陽離子交換層析法 21 9-5.等電聚焦分級分離儀 22 十、自製碳十八管柱去鹽 24 十一、奈米級液相層析電噴灑游離串聯式質譜 25 十二、資料分析 28 第三章 實驗結果與討論 30 一、樣品去鹽濃縮與蛋白質測定 30 二、不同分餾策略之鑑定散佈結果 30 2-1.酸性逆相層析法 31 2-2.鹼性逆相層析法 31 2-3.親水作用層析法 32 2-4.強陽離子交換層析法 32 2-5.等電聚焦分級分離儀 33 三、SCX與sIEF分析去鹽與未去鹽樣品比較 34 四、不同分餾策略之分離正交性 35 五、不同性質的胜肽分佈情形 36 六、分餾策略之互補性結果 38 第四章 結論 40 附圖 41 附表 88 參考文獻 100

    1. Watson, J.D., The human genome project: past, present, and future. Science, 1990. 248(4951): p. 44-9.
    2. Lander, E.S., et al., Initial sequencing and analysis of the human genome. Nature, 2001. 409(6822): p. 860-921.
    3. Wasinger, V.C., et al., Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995. 16(7): p. 1090-4.
    4. Edde, B., et al., Posttranslational glutamylation of alpha-tubulin. Science, 1990. 247(4938): p. 83-5.
    5. Fenn, J.B., et al., Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989. 246(4926): p. 64-71.
    6. Karas, M. and F. Hillenkamp, Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem, 1988. 60(20): p. 2299-301.
    7. Ashcroft, A.E., Protein and peptide identification: the role of mass spectrometry in proteomics. Nat Prod Rep, 2003. 20(2): p. 202-15.
    8. Domon, B. and R. Aebersold, Mass spectrometry and protein analysis. Science, 2006. 312(5771): p. 212-7.
    9. O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975. 250(10): p. 4007-21.
    10. Motoyama, A. and J.R. Yates, 3rd, Multidimensional LC separations in shotgun proteomics. Anal Chem, 2008. 80(19): p. 7187-93.
    11. McCormack, A.L., et al., Direct analysis and identification of proteins in mixtures by LC/MS/MS and database searching at the low-femtomole level. Anal Chem, 1997. 69(4): p. 767-76.
    12. Delmotte, N., et al., Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis. J Proteome Res, 2007. 6(11): p. 4363-73.
    13. Gan, C.S., et al., A Comparative Study of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) versus SCX-IMAC-Based Methods for Phosphopeptide Isolation/Enrichment. J Proteome Res, 2008. 7(11): p. 4869-4877.
    14. Manadas, B., et al., Comparative analysis of OFFGel, strong cation exchange with pH gradient, and RP at high pH for first-dimensional separation of peptides from a membrane-enriched protein fraction. Proteomics, 2009. 9(22): p. 5194-5198.
    15. Harrison, D.J., et al., Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical-Analysis System on a Chip. Science, 1993. 261(5123): p. 895-897.
    16. Horth, P., et al., Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Molecular & Cellular Proteomics, 2006. 5(10): p. 1968-1974.
    17. Heller, M., et al., Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma. Electrophoresis, 2005. 26(6): p. 1174-88.
    18. Ros, A., et al., Protein purification by Off-Gel electrophoresis. Proteomics, 2002. 2(2): p. 151-6.
    19. Fraterman, S., et al., Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle. Proteomics, 2007. 7(18): p. 3404-16.
    20. Kong, R.P., et al., Development of online high-/low-pH reversed-phase-reversed-phase two-dimensional liquid chromatography for shotgun proteomics: a reversed-phase-strong cation exchange-reversed-phase approach. Journal of Chromatography A, 2011. 1218(23): p. 3681-8.
    21. Wang, Y., et al., Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics, 2011. 11(10): p. 2019-26.
    22. Krishnan, S., et al., OFFgel-based multidimensional LC-MS/MS approach to the cataloguing of the human platelet proteome for an interactomic profile. Electrophoresis, 2011. 32(6-7): p. 686-95.
    23. Gilar, M., et al., Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. Journal of Separation Science, 2005. 28(14): p. 1694-1703.
    24. Ernoult, E., E. Gamelin, and C. Guette, Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation. Proteome Sci, 2008. 6: p. 27.
    25. Hubner, N.C., S. Ren, and M. Mann, Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis. Proteomics, 2008. 8(23-24): p. 4862-72.
    26. Cao, Z., et al., Systematic Comparison of Fractionation Methods for In-depth Analysis of Plasma Proteomes. J Proteome Res, 2012.
    27. Zhou, F., et al., Online nanoflow reversed phase-strong anion exchange-reversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms. Anal Chem, 2011. 83(18): p. 6996-7005.
    28. Lohaus, C., et al., Multidimensional chromatography: a powerful tool for the analysis of membrane proteins in mouse brain. J Proteome Res, 2007. 6(1): p. 105-13.
    29. Ficarro, S.B., et al., Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis. Mol Cell Proteomics, 2011. 10(11): p. O111 011064.
    30. Fang, Y., D.P. Robinson, and L.J. Foster, Quantitative Analysis of Proteome Coverage and Recovery Rates for Upstream Fractionation Methods in Proteomics. J Proteome Res, 2010. 9(4): p. 1902-1912.
    31. Boersema, P.J., et al., Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy. J Proteome Res, 2007. 6(3): p. 937-46.
    32. Hao, P., et al., Novel application of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) in shotgun proteomics: comprehensive profiling of rat kidney proteome. J Proteome Res, 2010. 9(7): p. 3520-6.
    33. Zarei, M., et al., Comparison of ERLIC-TiO2, HILIC-TiO2, and SCX-TiO2 for global phosphoproteomics approaches. J Proteome Res, 2011. 10(8): p. 3474-83.
    34. Murphy, R.E., M.R. Schure, and J.P. Foley, Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal Chem, 1998. 70(8): p. 1585-1594.
    35. Gilar, M., et al., Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem, 2005. 77(19): p. 6426-6434.
    36. Washburn, M.P., D. Wolters, and J.R. Yates, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology, 2001. 19(3): p. 242-247.
    37. Wolters, D.A., M.P. Washburn, and J.R. Yates, 3rd, An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem, 2001. 73(23): p. 5683-90.
    38. Wagner, Y., et al., Multidimensional nano-HPLC for analysis of protein complexes. J Am Soc Mass Spectrom, 2003. 14(9): p. 1003-11.
    39. Licklider, L.J., et al., Automation of nanoscale microcapillary liquid chromatography-tandem mass spectromentry with a vented column. Anal Chem, 2002. 74(13): p. 3076-3083.
    40. Vollmer, M., P. Horth, and E. Nagele, Optimization of two-dimensional off-line LC/MS separations to improve resolution of complex proteomic samples. Anal Chem, 2004. 76(17): p. 5180-5.
    41. http://brc.se.fju.edu.tw/protein/purify/isoelect.htm
    42. https://www.chem.agilent.com/Library/brochures/5990-5596EN.pdf
    43. Lam, H.T., et al., Modeling the isoelectric focusing of peptides in an OFFGEL multicompartment cell. J Proteome Res, 2007. 6(5): p. 1666-76.
    44. Baker, E.S., et al., An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies. J Proteome Res, 2010. 9(2): p. 997-1006.
    45. Ikonomou, M.G., A.T. Blades, and P. Kebarle, Electrospray Ion Spray - a Comparison of Mechanisms and Performance. Anal Chem, 1991. 63(18): p. 1989-1998.
    46. Dooley, K.C., Tandem mass spectrometry in the clinical chemistry laboratory. Clin Biochem, 2003. 36(6): p. 471-81.
    47. Yates, J.R., 3rd, et al., Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem, 1995. 67(8): p. 1426-36.
    48. Mortz, E., et al., Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases. Proc Natl Acad Sci U S A, 1996. 93(16): p. 8264-7.
    49. Alpert, A.J. and P.C. Andrews, Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J Chromatogr, 1988. 443: p. 85-96.
    50. Alpert, A.J., Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J Chromatogr, 1990. 499: p. 177-96.
    51. Bartha, A. and J. Stahlberg, Electrostatic Retention Model of Reversed-Phase Ion-Pair Chromatography. Journal of Chromatography A, 1994. 668(2): p. 255-284.
    52. Molnar, I. and C. Horvath, Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography. J Chromatogr, 1977. 142: p. 623-40.
    53. Nesvizhskii, A.I., et al., A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem, 2003. 75(17): p. 4646-58.
    54. Keller, A., et al., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem, 2002. 74(20): p. 5383-92.
    55. Kyte, J. and R.F. Doolittle, A simple method for displaying the hydropathic character of a protein. J Mol Biol, 1982. 157(1): p. 105-32.
    56. Gilar, M. and A. Jaworski, Retention behavior of peptides in hydrophilic-interaction chromatography. Journal of Chromatography A, 2011. 1218(49): p. 8890-6.
    57. Zhou, H., et al., A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis. Electrophoresis, 2007. 28(23): p. 4311-9.
    58. Peng, J., et al., Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res, 2003. 2(1): p. 43-50.
    59. Schley, C., et al., Proteome analysis of Myxococcus xanthus by off-line two-dimensional chromatographic separation using monolithic poly-(styrene-divinylbenzene) columns combined with ion-trap tandem mass spectrometry. J Proteome Res, 2006. 5(10): p. 2760-2768.
    60. Gilar, M., et al., Comparison of 1-D and 2-D LC MS/MS methods for proteomic analysis of human serum. Electrophoresis, 2009. 30(7): p. 1157-67.
    61. Slebos, R.J., et al., Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry. J Proteome Res, 2008. 7(12): p. 5286-94.
    62. Mihailova, A., et al., Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP. J Sep Sci, 2008. 31(3): p. 459-67.
    63. Toll, H., et al., Separation, detection, and identification of peptides by ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry at high and low pH. Journal of Chromatography A, 2005. 1079(1-2): p. 274-286.
    64. Waller, L.N., K. Shores, and D.R. Knapp, Shotgun proteomic analysis of cerebrospinal fluid using off-gel electrophoresis as the first-dimension separation. J Proteome Res, 2008. 7(10): p. 4577-4584.
    65. Chenau, J., et al., Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling. Proteome Sci, 2008. 6.

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