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 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.

Gel electrophoresis in combination with laser ablation–inductively coupled plasma mass spectrometry to quantify the interaction of cisplatin with human serum albumin

“…With the aim to provide a method that allows for detection and quantification of protein–metal adducts in gels, which are often used for the separation of a large number of proteins in complex matrices, we have used GE in combination with LA–ICP‐MS. For method development, we employed cisplatin and HSA whose reactions are well characterized . In order to be able to analyze gels by LA–ICP‐MS, a homogeneous, planar surface needs to be prepared.…”

“…This can be achieved by gel drying or through electroblotting of a nitrocellulose membrane. It has been shown that gel drying is the most efficient method and retains the quantitative features of GE . However, the gels need to be completely dry for efficient energy transfer .…”

“…Many of the interactions with proteins may have a significant impact on the structure of the protein and with the potential for metals to act as linking agents, forming covalently‐linked protein conjugates. In order for such modification to be studied, we and others are interested in developing new methods to elucidate the reactions occurring in complex matrices with many binding partners present .…”

Application of MeCAT-Click labeling for protein abundance characterization of E. coli after heat shock experiments

“…The ablation cell is a prototype design from D. Günther (ETH Zürich) which was already used for ablating electrophoretic gel and its design was altered by adding a second gas inlet opposite to the primary inlet [28]. Before the analysis, the system was warmed up and tuned on a daily basis by ablating glass standard (NIST 612, USA; line scans with 200 µm spot size, 10 µm s -1 scan rate, 20 Hz repetition rate and 100% laser energy).…”

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side‐by‐side

“…The corresponding 195 Pt images are shown as insets for both an unstained gel (A1, B1, C1) and a silver stained gel (A2, B2, C2). Left, inset: color scale for 195 Pt monitoring, common for all the images except for C1, which presents its own scale…”