title: Measuring site occupancy: a new perspective on cysteine oxidation.
abstract: Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass spectrometry. The method relies on differential reduction of "total" cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMT(TM) alkylation. Enrichment of iodoTMT(TM)-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH. We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response.
authors.affiliation: (1)University of Southern Denmark (Protein Research Group), Department of Biochemistry and Molecular Biology, Denmark.. Electronic address: adelinar@bmb.sdu.dk.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
authors.affiliation: (1)University of Southern Denmark (Protein Research Group), Department of Biochemistry and Molecular Biology, Denmark.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wojdyla
authors.affiliation: (1)University of Southern Denmark (Protein Research Group), Department of Biochemistry and Molecular Biology, Denmark.
authors.forename: James
authors.initials: J
authors.lastname: Williamson
authors.affiliation: (1)University of Southern Denmark (Protein Research Group), Department of Biochemistry and Molecular Biology, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Roepstorff