title: 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 (HPLC), 65% of the radioactivity copurified with the beta-subunit. Three adducted species were prominent in the ESI mass spectrum of the intact beta-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 beta-subunit resulted in a peptide mixture that, upon reversed-phase HPLC separation, provided several radiolabeled peptides. Using ESI-MS we identified these as the V91-101 and V102-122 peptides that represent the cysteine-containing peptides of the beta-subunit. These results provide definitive information on acrylamide-modified human hemoglobin and demonstrate that ESI-MS provides valuable structural information on chemically adducted proteins.
authors.affiliation: Biology and Chemistry Department, Pacific Northwest Laboratory, Richland, WA 99352.
authors.forename: D L
authors.initials: DL
authors.lastname: Springer
authors.forename: R J
authors.initials: RJ
authors.lastname: Bull
authors.forename: S C
authors.initials: SC
authors.lastname: Goheen
authors.forename: D M
authors.initials: DM
authors.lastname: Sylvester
authors.forename: C G
authors.initials: CG
authors.lastname: Edmonds
mesh_terms: D000178:Acrylamides / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006454:Hemoglobins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods
chemical_list: D000178:Acrylamides; D006454:Hemoglobins