title: Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.
abstract: Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.
authors: Proteomics Facility, College of Pharmacy, ‡the Howard Hughes Medical Institute, the Department of Molecular Genetics & Microbiology, and the Institute for Cellular and Molecular Biology, The University of Texas at Austin , Austin, Texas 78712, United States.
authors: Chia-Fang
authors: CF
authors: Lee
authors: Tanya T
authors: TT
authors: Paull
authors: Maria D
authors: MD
authors: Person
mesh_terms: D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012015:Reference Standards; D013454:Sulfoxides / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D016877:Oxidants; D020543:Proteome; D013454:Sulfoxides; D006861:Hydrogen Peroxide; C400529:PRMT5 protein, human; D011484:Protein-Arginine N-Methyltransferases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D003545:Cysteine
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