New perspectives in hemoglobin adducts analysis: selective digestion with calpain I

Background: Hemoglobin adducts are considered suitable biomarkers to evaluate human exposure to electrophile agents. One of the procedures used for the molecular dosimetry of hemoglobin adducts is the enzymatic digestion of the protein and the subsequent quantification of modified peptides by mass spectrometry. Due to the low level of modified hemoglobin peptides with respect to the corresponding unmodified ones, for the detection of adducts, samples must be enriched for them. Objectives: Within this item, hemoglobin was digested with Calpain I and the study was divided into two phases. Firstly we characterized proteolytic sites on pure hemoglobin, then we evaluate the ability of Calpain to discriminate between normal and alkylated globin chains.Methods: Digestions of pure hemoglobin sample and of an alkylated one with Calpain I at 200:1 and 25:1 weight ratios, for 1, 5 and 18 hrs were carried out. Results: By using a 200:1 weight ratio only peptides α(1-137), α(1-138) and β(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α- and β-chains. The identification of peptides obtained from the 25:1 digestions allowed the determination of internal secondary proteolytic sites, not reported in literature. In both cases, a non-negligible proteolysis was observed only when a reaction time of 18 hrs was used. Conclusions: Results showed a progressive enrichment of modified hemoglobin with respect to the unmodified one, particularly for the α-chain and represent a promising initial step in the development of a selective purification procedure to be applied for protein adducts analysis.