title: Reactive nitrogen oxide species-induced post-translational modifications in human hemoglobin and the association with cigarette smoking.
abstract: Nitric oxide (NO) is essential for normal physiology, but excessive production of NO during inflammatory processes can damage the neighboring tissues. Reactive nitrogen oxide species (RNOx), including peroxynitrite (ONOO(-)), are powerful nitrating agents. Biological protein nitration is involved in several disease states, including inflammatory diseases, and it is evident by detection of 3-nitrotyrosine (3NT) in inflamed tissues. In this study, we identified peroxynitrite-induced post-translational modifications (PTMs) in human hemoglobin by accurate mass measurement as well as by the MS(2) and MS(3) spectra. Nitration on Tyr-24, Tyr-42 (α-globin), and Tyr-130 (β-globin) as well as nitrosation on Tyr-24 (α-globin) were identified. Also characterized were oxidation of all three methionine residues, α-Met-32, α-Met-76, and β-Met-55 to the sulfoxide, as well as cysteine oxidation determined as sulfinic acid on α-Cys-104 and sulfonic acid on α-Cys-104, β-Cys-93, and β-Cys-112. These modifications are detected in hemoglobin freshly isolated from human blood and the extents of modifications were semiquantified relative to the reference peptides by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring (SRM) mode. The results showed a statistically significant positive correlation between cigarette smoking and the extents of tyrosine nitration at α-Tyr-24 and at α-Tyr-42. To our knowledge, this is the first report on identification and quantification of multiple PTMs in hemoglobin from human blood and association of a specific 3NT-containing peptide with cigarette smoking. This highly sensitive and specific assay only requires hemoglobin isolated from one drop (∼10 μL) of blood. Thus, measurement of these PTMs in hemoglobin might be feasible for assessing nitrative stress in vivo.
authors.affiliation: Department of Chemistry and Biochemistry, National Chung Cheng University, Ming-Hsiung, Chia-Yi, Taiwan. chehjc@ccu.edu.tw
authors.forename: Hauh-Jyun Candy
authors.initials: HJ
authors.lastname: Chen
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D030421:Peroxynitrous Acid / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D026361:Reactive Nitrogen Species / Q000737:chemistry*; D012907:Smoking; D021241:Spectrometry, Mass, Electrospray Ionization; D013441:Sulfinic Acids / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D006454:Hemoglobins; D010455:Peptides; D026361:Reactive Nitrogen Species; D013441:Sulfinic Acids; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D008715:Methionine; D003545:Cysteine