Proteomic approach for the analysis of acrylamide-hemoglobin adducts

The formation of adducts between acrylamide and hemoglobin in vitro was investigated by using mass spectrometric methodologies to identify the amino acid residues sensitive to alkylation. Liquid chromatography-electrospray ionisation mass spectrometry analysis of either intact or trypsin-digested α- and β-globin chains isolated from hemolysate samples incubated in vitro with acrylamide at different molecular ratios allowed us to identify Cys93 of β-globin as the most reactive site in hemoglobin, according to a Michael-type addition reaction between acrylamide and the sulphydryl group of cysteine. The only other reactive sites were Cys104 of α-globin and the N-terminal amino groups of both chains. The method developed, based on electrospray ionisation quadrupole time-of-flight tandem mass spectrometry analysis of intact globin chains was able to specifically detect low levels of adducts. In this way, rapid identification of alkylated portion of Hb was achieved to be potentially used as a biomarker for high-sensitivity biological monitoring.