Journal article
Mass-Spectrometry-Based Identification of Cross-Links in Proteins Exposed to Photo-Oxidation and Peroxyl Radicals Using O-18 Labeling and Optimized Tandem Mass Spectrometry Fragmentation
Protein cross-links are formed in regulated biochemical processes in many biological systems, but they are also generated inadvertently via the reactions of exogenous or endogenous oxidants. Site-specific identification and characterization of such cross-links is challenging, and the goal was, therefore, to develop mass-spectrometry-based approaches tailored for proteins subjected to oxidative challenges that also are applicable for the analysis of complex samples.
Using trypsin-mediated O-18 isotopic labeling, different types of data acquisition workflows, and designated database software tools, we successfully identified tyrosine-tyrosine, tyrosine-tryptophan, tyrosine-lysine, and histidine-lysine cross-links in proteins subjected to sensitizer-mediated photo-oxidation with rose bengal or chemical oxidation with peroxyl radicals generated from the water-soluble compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH).
Subsequently, AAPH was also applied to a protein extract from the Gram-positive bacterium Lactococcus lactic, demonstrating the feasibility to identify tyrosine-tyrosine, tyrosine-tryptophan, and tryptophan -tryptophan cross-linked peptides in a complex system. Different fragmentation techniques were evaluated, and it was observed that higher-energy collisional dissociation (HCD) resulted in a higher number of identified cross-link peptides, while electron transfer dissociation supplemented with HCD (EThcD) generally provides higher fragment ion coverage of the cross-linked peptides.
Language: | English |
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Publisher: | American Chemical Society |
Year: | 2018 |
Pages: | 2017-2027 |
ISSN: | 15353907 and 15353893 |
Types: | Journal article |
DOI: | 10.1021/acs.jproteome.7b00881 |
ORCIDs: | 0000-0002-5196-6919 , 0000-0002-6627-7518 and Svensson, Birte |
18O labeling AAPH Cross-linking ETD EThcD HCD LC−MS/MS Mass spectrometry Peroxyl radicals Photo-oxidation
Aptamers, Peptide Atto 488 maleimide Cross-Linking Reagents Fluoresceins Isotope Labeling LC-MS/MS O-18 labeling Oxidation-Reduction Peptides Peroxides Proteins SELEX Aptamer Technique Tandem Mass Spectrometry cross-linking mass spectrometry perhydroxyl radical peroxyl radicals photo-oxidation