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

Abstract

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. The aim of the study is to 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. Only the standard deviations (SD) of the ratios between alkylated globins with respect to the unmodified ones (αECH/α and bECH/b) were estimated because the aim of the study is to assess the ability of Calpain I to selectively digest the samples and, above all, to find the optimal digestion conditions and characterize obtained peptides. Results: by using a 200:1 weight ratio only peptides α(1-137), α(1-138) and b(1-142) were produced, pointing out that the first proteolytic sites are located at the C-terminus ends of both α-and b-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. Discussion: 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 to protein adducts analysis.