Artificial Oxidation of Cysteine Residues in Peroxiredoxin 6 Detected by Twodimensional Gel Electrophoresis and Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Abstract

Artificially oxidized cysteine residues in peroxiredoxin 6 (Prx6) were detected by electrospray interface capillary liquid chromatography-linear ion trap mass spectrometry after the preparation of two-dimensional gel electrophoresis (2D-GE). We used Prx6 as a model protein because it possesses only two cysteine residues at the 47th and 91st positions. The spot of Prx6 on 2D-GE undergoes a basic (isoelectric point, pI 6.6) to acidic (pI 6.2) shift by exposure to peroxide due to selective overoxidation of the active-site cysteine Cys-47 but not of Cys-91. However, we detected a tryptic peptide containing cysteine sulfonic acid at the 47th position from the basic spot and a peptide containing both oxidized Cys-47 and oxidized Cys-91 from the acidic spot of Prx6 after the separation by 2D-GE. We prepared two types of oxidized Prx6s: carrying oxidized Cys-47 (single oxidized Prx6), and other carrying both oxidized Cys-47 and Cys-91 (double oxidized Prx6). Using these oxidized Prx6s, the single oxidized Prx6 and double oxidized Prx6 migrated to pIs at 6.2 and 5.9, respectively. These results suggest that oxidized Cys-47 from the basic spot and oxidized Cys-91 from the acidic spot are generated by artificial oxidation during sample handling processes after isoelectric focusing of 2D-GE. Therefore, it is important to make sure of the origin of cysteine oxidation, if it is physiological or artificial, when an oxidized cysteine residue(s) is identified.

Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

“…Existing bottom-up proteomics strategies involve digestion of proteins either in solution or following one- or two-dimensional gel electrophoresis [ 17 ]. Oxidation is commonly encountered in cysteine, methionine, and tryptophan residues during gel electrophoresis and associated sample handling [ 18 – 20 ]. Whilst oxidation may result in reduced signal-to-noise due to the spread of signal over a number of mass channels, it does not prevent protein identification.…”

Housefly Pupae-Derived Antioxidant Peptides Exerting Neuroprotective Effects on Hydrogen Peroxide-Induced Oxidative Damage in PC12 Cells

“…Molecular weights of synthetic DR12 (1338.48 Da) and synthetic AR12 (1485.66 Da) were consistent with the theoretical masses of 1338.49 Da and 1485.66 Da, respectively. Some studies have reported that DR12 is a part of peroxiredoxin 6 (Prx6) structure (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53), which is an enzyme ubiquitously existing in life [31,32]. And several studies reported that AR12 is a part of heat shock proteins derived from human or dairy cows, respectively [33,34].…”

Bismuth-Carboxylate Ligand 1,3,6,8-Tetrakis(p-benzoic acid)pyrene Frameworks, Photophysical Properties, Biological Imaging, and Fluorescent Sensor for Biothiols

“…Abnormal levels of Cys, GSH, and Hcy are related to a number of diseases, which have effects on the normally physiological and pathological functions, such as AIDS, Alzheimer, angiocardiopathy, cancer, liver damage, and osteoporosis. , The analysis and detection of biothiols is significant for the prevention, research, and treatment of the abovementioned diseases and their biological functions. Compared with high-performance liquid chromatography, enzymatic methods, mass spectrometry, electrochemical assays, and Raman scattering, , fluorescence analysis methods , based on fluorescent probes have the advantages of simplicity, high selectivity, and short response time. More importantly, fluorescent probes are widely used in biomedicine, analytical chemistry, and chemical biology because they can carry out real-time monitoring and bio-imaging in organisms.…”