Mass-Spectrometry-Based Identification of Cross-Links in Proteins Exposed to Photo-Oxidation and Peroxyl Radicals Using ¹⁸O Labeling and Optimized Tandem Mass Spectrometry Fragmentation

Abstract

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 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 lactis, 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.

Detection, identification, and quantification of oxidative protein modifications

“…As the isotope analogues co-elute, a mass difference of 4 Da is observed for the linear peptides from the two 18 O atoms incorporated, but an 8-Da mass difference is detected for crosslinked peptides; this ϩ8-Da shift is diagnostic for the crosslinked species (Fig. 11) (41,262). This method has been used to identify His-His links in IgG 263 JBC REVIEWS: Detection of oxidative protein modifications links in glucose-6-phosphate dehydrogenase, RNase, and lysozyme (42,227,262).…”

UV oxidation of cyclic AMP receptor protein, a global bacterial gene regulator, decreases DNA binding and cleaves DNA at specific sites

“…products was determined on peptides generated by tryptic digestion as described previously [31][32][33] . Samples (150 µg protein) were prepared as described above, then subjected to buffer exchange into 100 mM ammonium bicarbonate buffer (ABC buffer) using spin filters (10 kDa cut-off).…”

“…Mass spectrometric analysis of cross-linked peptides. Analysis of cross-linked peptides by MS was performed as described previously 18,32 . Briefly, after tryptic peptides were digested in H 2 18 O-or H 2 16 O, they were subjected to solid-phase extraction on activated StageTip C18 reversed-phase discs, and peptides were then dried down (Speedvac concentrator, 3 mins), re-suspended in 10 μl H 2 18 O and H 2 16 O water, respectively, and mixed at a 1:1 ratio immediately prior to analysis.…”

“…Effects of UV irradiation on CRP-DNA complexes and isolated CRP assessed by gel mobility shift assays (A,B) and SDS-PAGE (C,D). Panel A: Gel mobility shift assay of CRP-cAMP complexes (50 µM cAMP) incubated in the dark, or exposed to UV (20 min exposure, λ max 310 nm, 30 nm band width, 6 W m −2 , normoxia), before addition to 32 P-labeled DNA in CRP binding buffer (see Materials and methods). DTT (1 mM), NaN 3 (100 mM) and mannitol (100 mM) were present as indicated.…”

Structural and functional changes in RNAse A originating from tyrosine and histidine cross-linking and oxidation induced by singlet oxygen and peroxyl radicals

“…Mass spectrometric analysis for the identification of cross-linked peptides was performed essentially as described previously [25,40]. Briefly, tryptic peptides digested in 18 O-and 16 Owater were mixed and immediately separated on an EASY nLC 100 chromatograph coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher).…”