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Characterization of sulfur mustard induced structural modifications in human hemoglobin by liquid chromatography-tandem mass spectrometry

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Author: Noort, D. · Verheij, E.R. · Hulst, A.G. · Jong, L.P.A. de · Benschop, H.P.
Type:article
Date:1996
Institution: Prins Maurits Laboratorium TNO TNO Voeding
Source:Chemical Research in Toxicology, 4, 9, 781-787
Identifier: 233355
doi: doi:10.1021/tx9502148
Keywords: Toxicology · globin · hemoglobin · mustard gas · trypsin · alkylation · article · blood · chemical warfare · human · liquid chromatography · protein modification · tandem mass spectrometry · Alkylation · Amino Acid Sequence · Carcinogens · Chemical Warfare Agents · Chromatography, High Pressure Liquid · Erythrocytes · Gas Chromatography-Mass Spectrometry · Hemoglobins · Humans · Molecular Sequence Data · Mustard Gas · Peptide Fragments · Spectrophotometry, Ultraviolet · Sulfur Radioisotopes · Trypsin

Abstract

In this paper we describe the use of tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to bis(2- chloroethyl) sulfide (sulfur mustard). Globin isolated from human whole blood which had been exposed to sulfur mustard was degraded with trypsin, and the digests were analyzed by micro LC/MS. Alkylated tryptic fragments (α-T1, α- T4, α-T6, α-T9, β-T1, β-T9, β-T10, β-T11, and β-T10-S-S-β-T12) could be tentatively assigned upon comparison with a digest from nonexposed globin. Subsequent tandem mass spectrometry of these peptides allowed unambiguous assignment of 5 specific modified residues: α-Val-1, α-His-20, β-Val-1, β-His-77, and β-His-97. The results demonstrate the usefulness of microbore LC in combination with tandem mass spectrometry for the structural determination of chemically modified peptides and proteins.