Person: Cañas Montalvo, Benito
Universidad Complutense de Madrid
Faculty / Institute
Now showing 1 - 6 of 6
PublicationEstrategias bioanalíticas en estudios metalómicos de fármacos de platino(Real Sociedad Española de Química, 2012) Gómez Gómez, M.Milagros; Moreno Gordaliza, Estefanía; Mena Fernández, María Luz; Palacios Corvillo, M. Antonia; Cañas Montalvo, BenitoEn este artículo se muestra el potencial que presentan las modernas estrategias bioanalíticas en estudios metalómicos de fármacos de platino. La combinación de técnicas de separación multidimensionales cromatográficas y/o electroforéticas con la espectrometría de masas atómica ICP-MS y molecular ESI-MS/MS se presenta como una valiosa alternativa en este tipo de estudios. PublicationOFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins(Elsevier, 2011) Mena, María Luz; Moreno Gordaliza, Estefanía; Moraleja San José, Irene; Cañas Montalvo, Benito; Gómez Gómez, M.MilagrosIn this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum–protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and postseparation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum–protein bond stability under the conditions studied. SDS-PAGE in the absence of -mercaptoethanol or dithiotreitol preserved the platinum–protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3–2.0 g were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated. PublicationNovel insights into the bottom-up mass spectrometry proteomics approach for the characterization of Pt-binding proteins: The insulin-cisplatin case study(Royal Society of Chemistry, 2010) Moreno Gordaliza, Estefanía; Cañas Montalvo, Benito; Palacios Corvillo, M.Antonia; Gómez Gómez, M.MilagrosThe characterization of the interaction of platinum drugs with proteins has been previously performed using bottom-up proteomics approaches (enzymatic digestion followed by MS analysis). Nevertheless, the study of the stability of the Pt–protein bonds along the whole process has been obviated for the moment. Herein the suitability of the treatments implied during enzymatic digestion of Pt–protein adducts has been evaluated, focusing on the stability of the Pt bonds. Insulin–cisplatin adducts were generated in vitro and separated from unreacted cisplatin by HPLC, the separation being checked by HPLC-ICP-MS. The chromatographically isolated Pt–insulin adducts have been proved to resist overnight digestion including treatment with Urea, DTT, IAA and trypsin in a Tris buffer. Direct analysis of the peptides generated by nESI-LIT MS allowed the determination of Pt-binding sites in insulin as: B Chain N-terminus, His5, His10, Cys7, Cys19 and A Chain Cys6, Cys7, Cys20. Results have been compared to a previous top-down approach, indicating that more complete information can be obtained with the bottom-up approach. Reactivity of free cysteines has been proved to prevail to N-donor groups, but when cysteines participate in disulfide bonds, their reactivity is comparable to N-donor sites (N-terminus, His). Preliminary results indicate that the use of High Intensity Focused Ultrasound for accelerating the enzymatic digestions is compatible with preserving Pt–protein bonds, allowing a reduction in the total digestion time to 5 min. Pt-containing peptides were fragmented and sequenced by CID, and results were compared with those obtained by the use of ETD, being CID spectra far more informative. PublicationTop-Down Mass Spectrometric Approach for the Full Characterization of Insulin-Cisplatin Adducts(ACS, 2009) Moreno Gordaliza, Estefanía; Cañas Montalvo, Benito; Palacios Corvillo, M. Antonia; Gómez Gómez, M.MilagrosThe interaction of the antitumor drug cisplatin with insulin was studied using a top-down mass spectrometric approach. In vitro incubations were prepared under acidic and physiological conditions at different insulin/cisplatin molar ratios for different incubation times. Size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICPMS) analysis enabled the specific detection of platinum containing species attributed to the binding of the drug to the protein. Further analysis through matrix-assisted laser desorption ionization-timeof-flight mass spectrometry (MALDI-TOF MS) and nanoelectrospray ionization mass spectrometry using a linear ion trap (nESI-LIT-MS) allowed the identification of platinated mono-, di-, and even triadducts in the incubations. Platinum binding sites were identified by CID-MSn as B chain N-terminus, His5, and probably His10 residues, which turned out to be the same, regardless of the incubation conditions. Evidence on the binding of Pt to B chain Cys7 was also observed. Working with the LIT zoom scan mode provides enough resolution to discern the isotopic pattern for both precursor and fragment ions, allowing the differentiation of platinumcontaining ions. The elucidation of platinum binding sites in a native protein through a top-down approach has been performed for the first time with this type of instrument. PublicationBiospeciation of tungsten in the serum of diabetic and healthy rats treated with the antidiabetic agent sodium tungstate(Elsevier, 2011-05-30) Gómez Gómez, M.Milagros; Rodríguez Fariñas, Nuria; Cañas Montalvo, Benito; Domínguez, Jorge; Guinovart, Joan; Cámara Rica, CarmenIt is known that oral administration of sodium tungstate preserves the pancreatic beta cell function in diabetic rats. Healthy and streptozotocin-induced diabetic rats were treated with sodium tungstate for one, three or six weeks, after which the species of W in serum, were analysed. An increase in serum W with treatment time was observed. After six weeks, the serum W concentration in diabetic rats (70 mg L−1) was about 4.6 times higher than in healthy specimens. This different behaviour was also observed for Cu accumulation, while the Zn pattern follows the contrary. The patterns observed in the retention of Cu and Zn may be attributable to a normalization of glycaemia. The speciation analysis of W was performed using 2D separations, including an immunoaffinity packing and a SEC (Size Exclusion Chromatography) column coupled to an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for elemental detection. Ultrafiltration data together with SEC-ICP-MS results proved that around 80% of serum W was bound to proteins, the diabetic rats registering a higher W content than their healthy counterparts. Most of the proteinbound W was due to a complex with albumin. An unknown protein with a molecular weight higher tan 100 kDa was also found to bind a small amount of W (about 2%). MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) analysis of the desalted and concentrated chromatographic fractions confirmed albumin as the main protein bound to tungstate in rat serum, while no binding to transferrin (Tf) was detected. The interaction between glutathione and W was also evaluated using standard solutions; however, the formation of complexes was not observed. The stability of the complexes between W and proteins when subjected to more stringent procedures, like those used in proteomic methodologies (denaturing with urea or SDS, boiling, sonication, acid media, reduction with -mercaptoethanol (BME) or DTT (dithiotreitol) and alkylation with iodoacetamide (IAA), was also evaluated. Our results indicate that the stability of the complexes between W and proteins is not too high enough to remain unaltered during protein separation by SDS–PAGE in denaturing and reducing conditions. However, the procedures for in-solution tryptic digestion and for ESI-MS analysis in MeOH/H2O/with 0.1% formic acid could be used for protein identification without large loss of binding between W and proteins. PublicationLA-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(RSC, 2012) Moreno Gordaliza, Estefanía; Esteban Fernández, Diego; Giesen, Charlotte; Lehmann, Karola; Lázaro, Alberto; Tejedor, Alberto; Scheler, Christian; Cañas Montalvo, Benito; Jakubowski, Norbert; Linscheid, Michael W.; Gómez Gómez, M.MilagrosA 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.