Characterization of Intact Hemoglobin and Oxaliplatin Interaction by Nanoelectrospray Ionization Tandem Mass Spectrometry

Abstract

Background: Mass spectrometric (MS) detection of intact hemoglobin (Hb) adducts presents considerable analytical challenges because of the noncovalent association of the 4 subunits of Hb, and MS characterization of the interaction of intact Hb with platinum drugs has not been reported. We developed a technique for detecting intact Hb and its drug adduct and studied the interactions between intact Hb and oxaliplatin. Methods: We incubated a series of mixtures of Hb and oxaliplatin at 37°C for 24 h or 5 days to investigate adduct formation. Blood samples from colorectal cancer patients undergoing oxaliplatin treatment were analyzed for novel adducts of intact Hb-oxaliplatin, which were characterized with nanoelectrospray ionization quadrupole time-of-flight MS. Results: Two intact Hb adducts, one with the whole oxaliplatin molecule and the other with oxaliplatin losing the oxalate ligand, were identified. Analysis of erythrocytes from the cancer patients provided direct evidence that oxaliplatin accumulated as Hb adducts in erythrocytes. A higher fraction (ϳ70%) of Hb was bound to oxaliplatin in erythrocytes from a patient who could not tolerate oxaliplatin treatment than in erythrocytes from another patient who benefited from this treatment. Conclusions:The nanoelectrospray tandem MS technique enabled determination of the intact Hb tetramer and its association with oxaliplatin. Hb-oxaliplatin ad-

Mass Spectrometry: A Tool for Enhanced Detection of Hemoglobin Variants

“…Evaluation of the number of substitutions of the ␣-and ␤-chain revealed that some of the most frequently possible variants were rarely found, such as in the ␣-chain Leu3 Gln (15 possible variants, 0 detected) and Ala3 Ser (21, 2), or in the ␤-chain Ala3 Ser (15, 1) and Ala3 Gly (15,2). On the other hand, some substitutions are frequently found, such as His3 Arg (10,8) or Lys3 Asn (11,11) in the ␣-chain or Ala3 Asp (14, 13) and Gly3 Asp (12,12) in the ␤-chain.…”

“…ESI or MALDI-TOF MS is now becoming a common auxiliary method applied in routine Hb analysis. The use of MS techniques has led to the discovery of more than 60 new mutations (14 ), and even the intact Hb tetramer can be analyzed using a nanoESI MS technique (15 ). Furthermore, MALDI-TOF MS is a highly sensitive method that enables the analysis of Hb chains from a single red blood cell (16 ).…”

Mass spectrometry study of hemoglobin‐oxaliplatin complexes in colorectal cancer patients and potential association with chemotherapeutic responses

“…The instrumental and experimental conditions have been described in detail in previous studies. [21][22][23][24][25] Horse heart myoglobin (Sigma-Aldrich, St. Louis, MO, USA) was used to externally calibrate the nanoESI-MS instrument. HPLC-grade water and ammonium acetate (Fisher Scientific, Fair Lawn, NJ, USA) were used throughout the experiments.…”

“…In each of these mass spectra, the three clusters of peaks correspond to the 19 erythrocytes from the healthy volunteer who was not under any platinum drug treatment or medication is characteristic of normal intact Hb. 25 The nanoESI mass spectra of the erythrocytes from the cancer patients undergoing oxaliplatin treatment (patients 3, 7, and 14, Figs (Figs. 4(B), 4(C), and 4(D)).…”

“…These techniques enabled in vitro studies of cisplatin, carboplatin, and oxaliplatin binding with intact hemoglobin (Hb) and also with its subunits. [21][22][23][24][25] These studies showed that heme groups were released from hemoglobin due to drug binding. 22 We present here an investigation into the formation of oxaliplatin-protein complexes in colorectal cancer patients who were undergoing oxaliplatin treatment.…”