A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML)

Abstract

Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD.

High-throughput endogenous measurement of S-nitrosylation in Alzheimer's disease using oxidized cysteine-selective cPILOT

“…For example, in a redox proteomic study using duplex ICAT reagents, because there are no more channels available for protein quantification, a separate experiment using isotopic dimethyl labeling is employed to provide protein abundance data 48 . Even in our OxcysDML approach, two experiments were performed to obtain total cysteine oxidation levels and protein abundance information based on cysteine-containing peptides 27 . Two, there is a large difference (roughly two to three orders of magnitude 49 ) between total peptide and SNO-modified peptide abundance.…”

“…Isobaric tagging methods such as iodoTMT, TMT, or the inclusion of iTRAQ 22 allow greater sample multiplexing capabilities (i.e., up to eight samples can be analyzed simultaneously). We recently developed a simple, straightforward, and robust approach to measure oxidized cysteine in global proteome experiments, through coupling on-resin capture of cysteines and isotopic dimethylation reactions 27 . This approach, called OxcysDML, could be readily modified to study SNO by exchanging the dithiothreitol reducing agent with ascorbate.…”

Proteomic approaches to quantify cysteine reversible modifications in aging and neurodegenerative diseases

“…To date, numerous approaches have been developed and applied in biological studies, e.g. oxidized isotopecoded affinity tag [37], oxidized isobaric tag for relative and absolute quantitation [45], SNO analysis by resin-assisted capture [36], oxidized multiple reaction monitoring [40], cysteine tandem mass tags and isobaric tag for relative and absolute quantification [48], and two approaches developed by our laboratory: oxidized cysteine-selective dimethylation (OxcysDML) [76] and oxidized cysteine-selective cPILOT (OxcyscPILOT) [77]. Compared with global protein quantification, more variables have to be considered for cysteine redox quantification, such as the modification type investigated, the number of sample multiplexing channels required, the incorporation of chemical mass tags, the enrichment method, amount of starting material, and method for stoichiometric quantitation.…”

“…This simple method is suitable for comparing cysteine modifications from two samples with desirable proteome coverage, and can reach up to 5-plex comparison using other formaldehyde/cyanoborohydride isotopomers [96]. Ox-cysDML has been applied to study the redox proteome of liver tissues from an AD mouse model [76], as discussed below.…”

“…Oxidized proteins involved in lipid metabolism could be linked with oxidative stress in AD liver [76]. 2016…”