LA-ICP-MS and nHPLC-ESI-LTQ-FT-MS/MS for the analysis of cisplatin–protein complexes separated by two dimensional gel electrophoresis in biological samples
LA-ICP-MS and nHPLC-ESI-LTQ-FT-MS/MS for the analysis of cisplatin–protein complexes separated by two dimensional gel electrophoresis in biological samples
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DOI：
10.1039/c2ja30016h
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发表时间：
2012-08
影响因子：
3.4
通讯作者：
E. Moreno-Gordaliza;D. Esteban-Fernández;Charlotte Giesen;Charlotte Giesen;K. Lehmann;A. Lázaro;A. Tejedor;C. Scheler;B. Cañas;N. Jakubowski;M. Linscheid;M. M. Gómez-Gómez-M.-M.-Gómez-Gómez-1399021175
中科院分区：
文献类型：
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作者：
E. Moreno-Gordaliza;D. Esteban-Fernández;Charlotte Giesen;Charlotte Giesen;K. Lehmann;A. Lázaro;A. Tejedor;C. Scheler;B. Cañas;N. Jakubowski;M. Linscheid;M. M. Gómez-Gómez-M.-M.-Gómez-Gómez-1399021175
已经开发了一种用于分析生物样品中的Pt-蛋白质复合物的方法，所述生物样品先前经受顺铂处理。采用凝胶电泳分离蛋白质，LA-ICP-（SF）-MS高灵敏度检测与Pt结合的蛋白质。含Pt的斑点用胰蛋白酶在凝胶中消化，产生的肽用nHPLC-ESI-LTQ-FT-MS/MS鉴定。染色和凝胶处理，然后在Pt上进行激光消融，使用与顺铂一起温育的标准蛋白质评价了蛋白质键的保存。在非还原条件下进行2-DE分离，然后进行考马斯亮蓝或银染色，对于Pt-蛋白复合物是合适的，从而实现生物样品中蛋白质的良好分离。直接LA-ICP-MS分析甘油处理的干凝胶Pt蛋白监测导致更好的灵敏度，更可靠的相对Pt信号和更简单，更耗时的方法相比，分析印迹膜。凝胶的消融允许处理凝胶中剩余的未消融材料中的Pt点的蛋白质鉴定，使得不必平行运行几个凝胶用于单独的Pt检测和蛋白质鉴定。通过使用这种方法，铂配位的蛋白质，如α-2-巨球蛋白，转铁蛋白，白蛋白或血红蛋白，从体内顺铂治疗的大鼠血清中检测后，nrSDS-PAGE分离。此外，第一个完整的LA-ICP-MS金属蛋白轮廓图在2-DE凝胶已经产生，在这种情况下，铂蛋白复合物在肾近端小管上皮细胞（RPTECs）与顺铂孵育的检测。在含Pt的斑点中鉴定了几种蛋白质，其可能与主要影响肾脏中该细胞类型的药物诱导的肾毒性有关。
A method for the analysis of Pt–protein complexes in biological samples, previously subjected to cisplatin treatment, has been developed. Proteins were separated by gel electrophoresis, and those bound to Pt were detected with high sensitivity by LA-ICP-(SF)-MS. Pt-containing spots were in-gel digested with trypsin, and the peptides produced identified using nHPLC-ESI-LTQ-FT-MS/MS. The influence of protein separation conditions, staining and gel processing prior to laser ablation on Pt–protein bonds preservation have been evaluated using standard proteins incubated with cisplatin. 2-DE separation under non-reducing conditions followed by either Coomassie blue brilliant or silver staining is appropriate for Pt–protein complexes, achieving a good separating resolution of the proteins in biological samples. Direct LA-ICP-MS analysis of glycerol-treated dried gels for Pt–protein monitoring resulted in better sensitivity, more reliable relative Pt signals and a simpler and less time-consuming approach compared to the analysis of blotted membranes. Ablation of gels allowed tackling protein identification of Pt-spots in the remaining non-ablated material in the gel, making it unnecessary to run several gels in parallel for separate Pt detection and protein identification. By using this approach, Pt coordinated to proteins, such as α-2-macroglobulin, transferrin, albumin or hemoglobin, was detected in the serum from a rat treated in vivo with cisplatin after nrSDS-PAGE separation. Furthermore, the first complete LA-ICP-MS metalloprotein contour map in a 2-DE gel has been produced, in this case for the detection of Pt–protein complexes in renal proximal tubule epithelial cells (RPTECs) incubated with cisplatin. Several proteins were identified in those spots containing Pt, which may have a connection with the drug-induced nephrotoxicity mainly affecting this cell type in the kidney.