title: Mass spectrometric studies of cisplatin-induced changes of hemoglobin.
abstract: This study reports on structural changes of hemoglobin (Hb) that were induced by cisplatin binding. Two techniques, nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoES-MS) and high-performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC/ICPMS), were developed to facilitate this study. Nanospray MS analyses of cisplatin and Hb reaction mixtures demonstrated that the ion at m/z 616.5, the heme group, increased with an increase of cisplatin concentration, indicating the loss of heme groups from the intact protein. This conclusion was also supported by the increase of cisplatin-alpha or -beta complex formation. The change of the Hb-bound Fe was further investigated by monitoring Fe signals using size-exclusion HPLC/ICPMS. After incubation with cisplatin at clinically relevant concentrations, under physiological conditions, the amount of Fe bound to Hb was reduced while formation of cisplatin-Hb complexes increased. Flow-injection ICPMS analysis of the Fe contents in the low molecular weight fraction (<3000 Da) of the reaction mixtures after size fractionation further demonstrated a corresponding increase of Fe with the increase of cisplatin concentrations. HPLC/ICPMS detected three Hb-cisplatin complexes, one of which eluted at the same retention time as Hb while the other two complexes eluted later than Hb. With clinically relevant concentrations of cisplatin (0.05-1.0 microM) and 10 microM of Hb, the concentrations of the Hb-cisplatin complexes were determined in the range 0.1-64 nM. These results, obtained from nanoES-MS, HPLC/ICPMS, and FIA-ICPMS, demonstrate that cisplatin binding to Hb resulted in the dissociation of the heme group from the intact protein.
authors.affiliation: Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 Canada.
authors.forename: Rupasri
authors.initials: R
authors.lastname: Mandal
authors.forename: Robyn
authors.initials: R
authors.lastname: Kalke
authors.forename: Xing-Fang
authors.initials: XF
authors.lastname: Li
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002945:Cisplatin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008832:Microchemistry / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006454:Hemoglobins; D006418:Heme; D002945:Cisplatin