S-Nitrosylation in Alzheimer’s Disease Using Oxidized Cysteine-Selective cPILOT

  • Ryan R. Dyer
  • Liqing Gu
  • Renã A. S. RobinsonEmail author
Part of the Neuromethods book series (NM, volume 127)


Cysteine S-nitrosylation (SNO) has important physiological roles related to maintaining protein activity, influencing protein conformation, and signaling apoptotic pathways. In Alzheimer’s disease (AD), SNO can exhibit neuroprotective effects through the inhibition of detrimental enzyme activity. However, in AD SNO is also implicated in mitochondrial dysfunction, neuronal loss, impaired metabolism, and protein misfolding and aggregation. In order to better understand the beneficial and detrimental effects of SNO, SNO sites in disease states must first be characterized. This requires robust analytical methods for the identification and quantification of SNO-modified proteins. Additionally, detection of endogenous SNO modifications requires highly sensitive methods, as this modification exists in low concentrations. Here we describe oxidized cysteine-selective combined precursor isotopic labeling and isobaric tagging (OxcyscPILOT), a high-throughput method for the identification and quantification of endogenous peptide SNO sites relative to the entire cysteine proteome. Combining selective reduction and enrichment of SNO peptides with the cPILOT methodology extends multiplexing capabilities to 12 samples with TMT6 reagents, 20 samples with TMT10 reagents, and potentially greater with custom isobaric tags. Our goal is to provide the reader with step-by-step instructions to perform OxcyscPILOT through demonstration with mouse brain tissues.

Key words

Cysteine Redox proteomics S-Nitrosylation Alzheimer’s disease Multiplexing 



The authors would like to acknowledge the National Institutes of Health for financial support (R01 GM 117191-01).


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ryan R. Dyer
    • 1
  • Liqing Gu
    • 1
  • Renã A. S. Robinson
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of PittsburghPittsburghUSA

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