Analysis of Chlorination, Nitration, and Nitrosylation of Tyrosine and Oxidation of Methionine and Cysteine in Hemoglobin from Type 2 Diabetes Mellitus Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

The post-translational modification (PTM) of proteins by endogenous reactive chlorine, nitrogen, and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications is higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of 12 sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at alpha-Tyr-24, nitration at alpha-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n = 19) than in nondiabetic individuals (n = 18). After excluding the factor of smoking, chlorination at alpha-Tyr-24, nitration at alpha-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n = 12) than in normal nonsmoking subjects (n = 11). Multiple regression analysis performed on the combined effect of age, body-mass index (BMI), and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination at alpha-Tyr-24 in nonsmokers. In addition, age contributes-to oxidation at alpha-Met-32 significantly in all subjects and in nonsmokers. These results suggest the potential of using chlorination at alpha-Tyr-24-containing peptide to evaluate protein damage in nonsmoking type 2 diabetes mellitus.