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

Abstract

Reversible cysteine modifications play important physiological roles such as modulating enzymatic catalysis, maintaining redox homeostasis and conducting cellular signaling. These roles can be critical in the context of disease. Oxidative modifications such as S-nitrosylation (SNO) are signatures of neurodestruction in conditions of oxidative stress however are also indicators of neuroprotection and normal signaling in cellular environments with low concentrations of reactive oxygen and nitrogen species. SNO is a dynamic and low abundance modification and requires sensitive and selective analytical methods for its detection in biological tissues. Here we present an enhanced multiplexing strategy to study SNO in complex mixtures arising from tissues. This method, termed oxidized cysteine-selective cPILOT (OxcyscPILOT), allows simultaneous analysis of SNO-modified peptides in 12 samples. OxcyscPILOT has three primary steps: (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing SNO on a solid phase resin, and (3) isotopic labeling and isobaric tagging of enriched peptides on the solid phase resin. This approach offers the advantage of allowing total protein abundance levels to be measured simultaneously with endogenous SNO levels and measurement of SNO levels across four biological replicates in a single analysis. Furthermore, the relative amount of SNO on a specific cysteine site can also be determined. A well-known model of Alzheimer’s disease, the APP/PS-1 transgenic mouse model, was selected for demonstration of the method as several SNO-modified proteins have previously been reported in brain and synaptosomes from AD subjects. OxcyscPILOT analysis resulted in identification of 138 SNO-modified cysteines in brain homogenates that corresponds to 135 proteins. Many of these SNO-modified proteins were only present in wild-type or AD mice, whereas 93 proteins had SNO signals in both WT and AD. Pathway analysis links SNO-modified proteins to various biological pathways especially metabolism and signal transduction, consistent with previous reports in the literature. The OxcyscPILOT strategy provides enhanced multiplexing capability to current redox proteomics methods to study oxidative modifications of cysteine.

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

“…By combining isotopic and isobaric labeling, highly multiplexed quantification can be achieved with RAC for redox proteomics. 108–110 The covalent capture and immobilization of free thiol-containing protein/peptides using the RAC approach allows for more stringent washing to further reduce contaminants and non-specific binding. Isobaric labeling coupled with RAC allows for multiplexed quantification and stoichiometric measurement for multiple Cys redox PTMs, including SNO, SSG and total thiol oxidation 66, 69, 73, 79, 80 .…”

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

“…Alzheimer's disease is a neurodegenerative disorder characterized by neurofibrillary tangles, senile plaques, loss of synapses among other pathological hallmarks and has well‐known oxidative stress in brain , plasma , heart , spleen , and liver tissues. Similar to aging, cysteine reversible modifications are also implicated in AD pathogenesis.…”

“…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.…”

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…Comprehensive and site-specific analysis of protein modifications is normally beyond the reach of conventional biochemistry methods. Modern MS technology provides a unique opportunity to globally and site-specifically characterize protein modifications (Ficarro et al, 2002;Peng et al, 2003;Trinidad et al, 2006;Siuti and Kelleher, 2007;Witze et al, 2007;Yates et al, 2009;Mertins et al, 2013;Nwosu et al, 2013;Huang et al, 2015a;Ludwig et al, 2015;Gu and Robinson, 2016;Lossl et al, 2016;Yue et al, 2016;Cao et al, 2017;Simithy et al, 2017;Hogrebe et al, 2018;Wu et al, 2018). However, it is still extraordinarily challenging to globally analyze them because of the low abundance of many modified proteins, sub-stoichiometry of protein modifications, and the complexity of biological samples.…”