title: Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry.
abstract: Dried blood spot (DBS) is an emerging microsampling technique for the bioanalysis of small molecules, including fatty acids, metabolites, drugs, and toxicants. DBS offers many advantages as a sample format including easy sample collection and cheap sample shipment. Hemoglobin adducts have been recognized as a suitable biomarker for monitoring chemical exposure. We previously reported that certain modified peptides in hemoglobin derived from reactive chlorine, nitrogen, and oxygen species are associated with factors including smoking, diabetes mellitus, and aging. However, the stability of these oxidation-induced modifications of hemoglobin remains unknown and whether they can be formed artifactually during storage of DBS. To answer these questions, globin extracted from the DBS cards was analyzed, and the stability of the modifications was evaluated. After storage of the DBS cards at 4 °C or room temperature up to 7 weeks, we isolated globin from a quarter of the spot every week. The extents of 11 sites and types of post-translational modifications (PTMs), including nitration and nitrosylation of tyrosine and oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) using selected reaction monitoring. The extents of all these PTMs are stable within 14 days when stored on DBS at room temperature and at 4 °C, while those from direct extraction of fresh blood are stable for at least 8 weeks when stored as an aqueous solution at -20 °C. Extraction of globin from a DBS card is of particular importance for hemolytic blood samples. To our knowledge, this is the first report on the stability of oxidative modifications of hemoglobin on DBSs, which are stable for 14 days under ambient conditions (room temperature, in air). Therefore, it is feasible and convenient to analyze these hemoglobin modifications from DBSs in studies involving large populations.
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University , 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
authors.forename: Hauh-Jyun Candy
authors.identifier: 0000-0003-1869-4779
authors.initials: HC
authors.lastname: Chen
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University , 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
authors.forename: Chih-Huang
authors.initials: CH
authors.lastname: Fan
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University , 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
authors.forename: Ya-Fen
authors.initials: YF
authors.lastname: Yang
mesh_terms: D001769:Blood; D002853:Chromatography, Liquid / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D026361:Reactive Nitrogen Species / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006454:Hemoglobins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species