Combining TBP-based rOFFGEL-IEF with FASP and nLC-ESI-LTQ-MS/MS for the analysis of cisplatin-binding proteins in rat kidney.
In this work, a methodology based on a reducing IEF separation in combination with a FASP tryptic digestion able to maintain the integrity of cisplatin-protein complexes has been developed. The method is based on OFFGEL-IEF under conditions provided by the thiol-free reducing agent TBP, which allowed the separation of cisplatin-binding proteins in liquid fractions. The FASP procedure is applied as an intermediate stage between the IEF separation and MS analysis where the proteins are retained and concentrated in a commercially available ultrafiltration device. The filter unit acts as a proteomic reactor for detergent removal, buffer exchange, chemical modification (reduction and alkylation) and protein digestion. Finally, purified peptides are recovered by centrifugation. This procedure provides efficiencies comparable to standard in-solution digestion and the risk of platinum-complexes loss is minimized due to the fact that reagents employed along the process are subsequently eliminated before the following step. The stability of platinum-protein complexes under the FASP tryptic digestion, either using TBP or DTT as reducing agents, was maintained, allowing the identification of several platinum-containing peptides from cisplatin-HSA. This methodology was applied to the separation of platinum-enriched protein fractions obtained by SEC-ICP-MS in a kidney tissue extract from a rat treated with cisplatin, followed by further identification by nLC-ESI-LTQ-MS/MS after FASP tryptic digestion of selected platinum-containing liquid fractions.
Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. Electronic address: irenemoraleja@ucm.es.
I
I
Moraleja
Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. Electronic address: estefania.moreno@quim.ucm.es.
E
E
Moreno-Gordaliza
Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. Electronic address: mariluz@ucm.es.
M L
ML
Mena
Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. Electronic address: mmgomez@ucm.es.
M M
MM
Gómez-Gómez
D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods*; D002945:Cisplatin / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000970:Antineoplastic Agents; D011506:Proteins; D002945:Cisplatin
2-DE; CA; CYT C; DTT; FASP; HSA; IAA; ICP-MS; IEF; IPG; Kidney tissues; LA-ICP-MS; MYO; OFFGEL-IEF; Platinum-binding proteins; RPTECs; SEC–ICP-MS; TBP; TF; carbonic anhydrase; cytochrome c; dithiothreitol; filter-aided sample preparation; human apo-transferrin; human serum albumin; immobilized pH gradient; iodoacetamide; isoelectric focusing; laser ablation-inductively coupled plasma mass spectrometry; myoglobin; nLC–ESI-LTQ-MS/MS; nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry; non-reducing OFFGEL-IEF; nrOFFGEL-IEF; rOFFGEL-IEF; reducing OFFGEL-IEF; renal proximal tubule epithelial cells; size exclusion chromatography–inductively coupled plasma-mass spectrometry; tributylphosphine; two-dimensional gel electrophoresis