Electrospray ionization mass spectrometric characterization of acrylamide adducts to hemoglobin.
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.
Springer
D L
DL
Biology and Chemistry Department, Pacific Northwest Laboratory, Richland, WA 99352.
Bull
R J
RJ
Goheen
S C
SC
Sylvester
D M
DM
Edmonds
C G
CG
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
D000178:Acrylamides; D006454:Hemoglobins