Issue 9, 2012

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

Abstract

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.

Graphical abstract: 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

Supplementary files

Article information

Article type
Paper
Submitted
19 Jan 2012
Accepted
30 May 2012
First published
30 May 2012

J. Anal. At. Spectrom., 2012,27, 1474-1483

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

E. Moreno-Gordaliza, D. Esteban-Fernández, C. Giesen, K. Lehmann, A. Lázaro, A. Tejedor, C. Scheler, B. Cañas, N. Jakubowski, M. W. Linscheid and M. M. Gómez-Gómez, J. Anal. At. Spectrom., 2012, 27, 1474 DOI: 10.1039/C2JA30016H

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements