RT Journal Article
T1 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
A1 Moreno Gordaliza, María Estefanía
A1 Esteban Fernández, Diego
A1 Giesen, Charlotte
A1 Lehmann, Karola
A1 Lázaro Fernández, Alberto
A1 Tejedor Jorge, Alberto
A1 Scheler, Christian
A1 Cañas Montalvo, Benito
A1 Jakubowski, Norbert
A1 Linscheid, Michael W.
A1 Gómez Gómez, María Milagros
AB 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 timeconsuming 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 a-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.
PB RSC
SN 0267-9477
YR 2012
FD 2012
LK https://hdl.handle.net/20.500.14352/42740
UL https://hdl.handle.net/20.500.14352/42740
LA eng
NO Moreno Gordaliza, E., Esteban Fernández, D., Giesen, C. et al. «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». Journal of Analytical Atomic Spectrometry, vol. 27, n.o 9, 2012, p. 1474. DOI.org (Crossref), https://doi.org/10.1039/c2ja30016h.
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