title: Mass Spectrometric Analysis of Glyoxal and Methylglyoxal-Induced Modifications in Human Hemoglobin from Poorly Controlled Type 2 Diabetes Mellitus Patients.
abstract: Glyoxal and methylglyoxal are oxoaldehydes derived from the degradation of glucose-protein conjugates and from lipid peroxidation, and they are also present in the environment. This study investigated the site-specific reaction of glyoxal and methylglyoxal with the amino acid residues on human hemoglobin using a shot-gun proteomic approach with nanoflow liquid chromatography/nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). In human hemoglobin incubated with glyoxal, modification on 8 different sites, including lysine residues at α-Lys-11, α-Lys-16, α-Lys-56, β-Lys-17, β-Lys-66, β-Lys-144, and arginine residues at α-Arg-92 and β-Arg-30, was observed using a data-dependent scan. In methylglyoxal-treated hemoglobin, there were specific residues, namely, α-Arg-92, β-Lys-66, β-Arg-30, and β-Lys-144, forming carboxyethylation as well as the dehydrated product hydroimidazolone at α-Arg-92 and β-Arg-30. These lysine and arginine modifications were confirmed by accurate mass measurement and the MS(2) and MS(3) spectra. The most intensive signal of each modified peptide was used as the precursor ion to perform the product ion scan. The relative extent of modifications was semiquantified simultaneously relative to the native reference peptide by nanoLC-NSI/MS/MS under the selected reaction monitoring (SRM) mode. The extent of these modifications increased dose-dependently with increasing concentrations of glyoxal or methylglyoxal. Six out of the eight modifications induced by glyoxal and three out of the six modifications induced by methylglyoxal were detected in hemoglobin freshly isolated from human blood samples. The relative extent of modification of these post-translational modifications was quantified in poorly controlled type 2 diabetes mellitus patients (n = 20) and in nondiabetic control subjects (n = 21). The results show that the carboxymethylated peptides at α-Lys-16, α-Arg-92, β-Lys-17, β-Lys-66, and the peptide at α-Arg-92 with methylglyoxal-derived hydroimidazolone are significantly higher in diabetic patients than in normal individuals (p value <0.05). This report identified and quantified glyoxal- and methylglyoxal-modified hemoglobin peptides in humans and revealed the association of the extent of modifications at specific sites with T2DM. Only one drop (10 μL) of fresh blood is needed for this assay, and only an equivalent of 1 μg of hemoglobin was analyzed by the nanoLC-NSI/MS/MS-SRM system. These results suggest the potential use of these specific post-translational modifications in hemoglobin as feasible biomarker candidates to assess protein damage induced by glyoxal and methylglyoxal.
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.initials: HJ
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: Yu-Chin
authors.initials: YC
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: Chiung-Fong
authors.initials: CF
authors.lastname: Hsiao
authors.affiliation: Buddhist Dalin Tzu Chi General Hospital , No.2, Minsheng Road, Dalin, Chia-Yi 622, Taiwan.
authors.forename: Pin-Fan
authors.initials: PF
authors.lastname: Chen
mesh_terms: D003924:Diabetes Mellitus, Type 2; D005260:Female; D006037:Glyoxal / Q000737:chemistry / Q000633:toxicity*; D006454:Hemoglobins / Q000737:chemistry* / Q000187:drug effects*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000032:analysis / Q000097:blood; D011765:Pyruvaldehyde / Q000737:chemistry / Q000633:toxicity*; D012015:Reference Standards; D012907:Smoking; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D006454:Hemoglobins; D010455:Peptides; D006037:Glyoxal; D011765:Pyruvaldehyde