Identification of biomarkers indicating cellular changes after treatment of neuronal cells with the C3 exoenzyme from Clostridium botulinum using the iTRAQ protocol and LC-MS/MS analysis
Identification of biomarkers indicating cellular changes after treatment of neuronal cells with the C3 exoenzyme from Clostridium botulinum using the iTRAQ protocol and LC-MS/MS analysis
复制标题
DOI：
10.1016/j.jchromb.2008.12.005
复制
发表时间：
2009-05-01
影响因子：
3
通讯作者：
Pich, A.
中科院分区：
文献类型：
--
作者：
Muetzelburg, M. V.;Hofmann, F.;Pich, A.
蛋白质组学方法用于鉴定生物标志物，监测细胞内的病理变化和更好的疾病诊断。蛋白质稳态的可比变化也发生在分化细胞中，蛋白质组学技术应该适合于鉴定指示细胞发育的不同步骤的生物标志物。来自肉毒梭菌的C3胞外酶（C3 bot）使Rho GTP酶失活并诱导细胞形态学变化，如细胞变圆和神经突生长[G. Ahnert-Hilger，M. Holtje，G.格罗斯湾皮克特角穆克，B。Nixdorf-Bergweiler，P. Boquet，F.霍夫曼岛只是神经化学杂志90（2004）9]。为了进一步研究这些观察结果，选择了比较蛋白质组学方法来阐明C3 bot在神经母细胞瘤细胞系模型SH-SY 5 Y中的作用。用于生物标志物检测的筛选方法基于稳定同位素方法同量异位素标记用于相对和绝对定量（iTRAQ）。消化C3 bot处理的和未处理的细胞的蛋白质，并通过iTRAQ试剂标记肽，合并，并通过二维纳米HPLC系统分离。在MALDI-TOF/TOF质谱仪中进行肽分析。通过MS/MS光谱分析完成肽及其相应蛋白的鉴定和定量。总体而言，五次重复测量鉴定了355种不同的蛋白质，其中235种可用于定量。C3 bot改变了55种蛋白质的浓度（至少1.3倍），并且几种蛋白质被鉴定为可能的生物标志物候选物，表明C3 bot引起的细胞变化。(C)2008 Elsevier B. V.保留所有权利。
Proteomic approaches are used to identify biomarkers, to monitor pathological changes inside of cells and for a better diseases diagnosis. Comparable changes in protein homeostasis also occur in differentiating cells and proteomic techniques should be suitable to identify biomarkers that indicate different steps of cellular development. The C3 exoenzyme from Clostridium botulinum (C3bot) inactivates Rho GTPases and induces morphological cellular changes like cell rounding and neurite outgrowth [G. Ahnert-Hilger, M. Holtje, G. Grosse, G. Pickert, C. Mucke, B. Nixdorf-Bergweiler, P. Boquet, F. Hofmann, I. just, J. Neurochem. 90 (2004) 9]. To investigate these observations further a comparative proteomic approach has been chosen to elucidate C3bot effects in the neuroblastoma cell line model SH-SY5Y. The screening method applied for biomarker detection was based on the stable isotope approach isobaric tagging for relative and absolute quantification (iTRAQ). Proteins of C3bot-treated and untreated cells were digested and peptides were labeled by the iTRAQ reagent, combined, and separated by means of a two-dimensional nano-HPLC system. Peptide analysis was performed in a MALDI-TOF/TOF mass spectrometer. Identification and quantification of peptides and their corresponding proteins were accomplished by MS/MS spectra analysis. Overall, five replicate measurements identified 355 different proteins of which 235 were accessible for quantification. C3bot altered the concentration of 55 proteins (at least 1.3-fold) and several proteins were identified as possible biomarker candidates that indicate C3bot-incluced cellular changes. (C) 2008 Elsevier B.V. All rights reserved.