Electrospray ionization mass spectrometric characterization of acrylamide adducts to hemoglobin

Abstract

The most common procedure to identify hemoglobin adducts has been to cleave the adducts from the protein and characterize the adducting species, by, for example, derivatization and gas chromatography/mass spectrometry. To extend these approaches we used electrospray ionization mass spectrometry (ESI‐MS) to characterize adducted hemoglobin. For this we incubated [¹⁴C]acrylamide with the purified human hemoglobin (type A₀) under conditions that yielded high adduct levels. When the hemoglobin was separated by reversed‐phase high‐performance liquid chromatography (HPLO, 65% of the radioactivity copurified with the β‐subunit. Three adducted species were prominent in the ESI mass spectrum of the intact β‐subunit, indicating acrylamide adduction (i.e., mass increase of 71 Da) and two additional unidentified moieties with mass increments of 102 and 135 Da. Endoproteinase Glu‐C digestion of the adducted β‐subunit resulted in a peptide mixture that, upon reversed‐phase HPLC separation, provided several radiolabeled peptides.