人神经母细胞瘤的磷酸化膜蛋白质组分析

侯春彦; 马俊锋; 陶定银; 张丽华; 梁振; 张玉奎

doi:10.3724/SP.J.1123.2011.00851

化学进展 ›› :851-856 DOI: 10.3724/SP.J.1123.2011.00851

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人神经母细胞瘤的磷酸化膜蛋白质组分析

^1,2

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Phosphorylated membrane proteome analysis of human neuroblastoma SH-SY5Y cell line

HOU Chunyan^1,2, MA Junfeng^1,2, TAO Dingyin^1,2, ZHANG Lihua¹, LIANG Zhen¹, ZHANG Yukui¹

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摘要

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱（Ti⁴⁺-IMAC）整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1mol/L NaCl和0.1mol/L Na₂CO₃顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti⁴⁺-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。

Abstract

A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti⁴⁺ affinity chromatography(Ti⁴⁺-IMAC)was proposed,and applied in the global profiling of phosphorylated membrane proteome of neuroblastoma SH-SY5Y cells. The fraction of membrane proteins was extracted by ultra speed centrifuge,followed by wash-lng with 1 mol/L sodium chloride and 0.1 mol/L sodium carbonate. For digestion,chymotrypsin and pepsin with broader specificity were used as complementary enzymes to trypsin. The phosphopeptides were then selectively enriched by monolithic Ti⁴⁺-IMAC column,and analyzed by nanoflow high performance liquid chromatography and mass spectrometry. A total of 43 phosphoproteins were identified,among which 14 proteins were located on the membrane. All these results demonstrated that the proposed strategy might be promising to promote the in-depth study of neuroblastoma and discover the candidate biomarkers.

关键词

人神经母细胞瘤 / 磷酸化肽富集 / 膜蛋白质 / 多酶酶解 / 固定化钛离子亲和色谱 / 有机-无机杂化硅胶整体材料

Key words

human neuroblastoma / phosphopeptide enrichment / membrane protein / multienzyme digestion / immobilized Ti⁴⁺ affinity chromatography / organic-inorganic hybrid silica monolith

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侯春彦, 马俊锋, 陶定银, 张丽华, 梁振, 张玉奎. 人神经母细胞瘤的磷酸化膜蛋白质组分析[J]. 化学进展, , (): 851-856 DOI:10.3724/SP.J.1123.2011.00851

HOU Chunyan, MA Junfeng, TAO Dingyin, ZHANG Lihua, LIANG Zhen, ZHANG Yukui. Phosphorylated membrane proteome analysis of human neuroblastoma SH-SY5Y cell line[J]. Progress in Chemistry 851-856 DOI:10.3724/SP.J.1123.2011.00851

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参考文献

原文顺序 | 出版日期 | 本文引用

[1] Garcia J, Faca V, Jarzembowski J, et al. J Proteome Res, 2009, 8(8):3791

[2] Hsu W M, Lee H, Juan H F, et al. Clin Cancer Res, 2008, 14(19):6237

[3] Shin B K, Wang H, Yim A M, et al. J Biol Chem, 2003,278(9):7607

[4] Zhang Y, Qin H Q, Wu R A, et al. Chinese Journal of Chromatography,2010, 28(2):123

[5] Haque N, Gong C X, Sengupta A, et al. Brain Res Mol Brain Res, 2004, 129(1):163

[6] Blonder J, Goshe M B, Moore R J, et al. J Proteome Res, 2002, 1(4):351

[7] Ye X Y, Johann D Jr, Hakami R M, et al. J Proteomics, 2009, 73(1):112

[8] Hou C Y, Ma J F, Tao D Y, et al. J Proteome Res, 2010, 9(8):4093

[9] Bhatia V N, Perlinan D H, Costello C E, et al. Anal Chem,2009, 81(23):9819

[10] Tanaka T, Iqbal K, Trenkner E, et al. FEBS Lett, 1995, 360(1):5

[11] Garcia-Perez J, Avila J, Diaz-Nido J, et al. J Neurosci Res, 1998,52(4):445

[12] Sanchez S, Sayas C L, Lim F, et al. J Neurochem, 2001, 78(3):468

[13] de la Monte S M, Chen G J, Rivera E, et al. Cell Mol Life Sci, 2003,60(12):2679

[14] Lee S Y, Kim J W, Jeong M H, et al. FEBS Lett, 2008,582(19):2826

[15] Kumar H R, Zhong X L, Hoelz D J, et al. Pediatr Surg Int, 2008,24(11):1229

[16] Okada H, Zhang W, Peterhoff C, et al. Faseb J, 2010, 24(8):2783

[17] Tomonaga T, Levens D. J Biol Chem, 1995,270(9):4875

[18] Michelotti E F, Michelotti G A, Aronsohn A I, et al. Mol Cell Biol, 1996, 16(5):2350

[19] Choi H S, Song K Y, Hwang C K, et al. Mol Cell Proteomics, 2008,7(8):1517

[20] Beaudry H, Gendron L, Guimond M O, et al. Endocrinology, 2006, 147(9):4263