Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Current Proteomic Approaches Applied to Brain Function

Part of the book series: Neuromethods ((NM,volume 127))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Butterfield DA et al (2014) Mass spectrometry and redox proteomics: applications in disease. Mass Spectrom Rev 33(4):277–301

    Article  CAS  PubMed  Google Scholar 

  2. Lennicke C et al (2016) Redox proteomics: Methods for the identification and enrichment of redox-modified proteins and their applications. Proteomics 16(2):197–213

    Article  CAS  PubMed  Google Scholar 

  3. Foster MW, Hess DT, Stamler JS (2009) Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med 15(9):391–404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Verrastro I et al (2015) Mass spectrometry-based methods for identifying oxidized proteins in disease: advances and challenges. Biomol Ther 5(2):378–411

    CAS  Google Scholar 

  5. Couvertier SM, Zhou Y, Weerapana E (2014) Chemical-proteomic strategies to investigate cysteine posttranslational modifications. Biochim Biophys Acta 1844(12):2315–2330

    Article  CAS  PubMed  Google Scholar 

  6. García-Santamarina S, Boronat S, Hidalgo E (2014) Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction. Biochemistry 53(16):2560–2580

    Article  PubMed  Google Scholar 

  7. Gu L, Robinson RA (2016) Proteomic approaches to quantify cysteine reversible modifications in aging and neurodegenerative diseases. Proteomics Clin Appl 10(12):1159–1177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Meckler X et al (2010) Reduced Alzheimer’s disease β-amyloid deposition in transgenic mice expressing S-palmitoylation-deficient APH1aL and nicastrin. J Neurosci 30(48):6160–16169

    Article  Google Scholar 

  9. López-Sánchez LM, López-Pedrera C, Rodríguez-Ariza A (2014) Proteomic approaches to evaluate protein s-nitrosylation in disease. Mass Spectrom Rev 33(1):7–20

    Article  PubMed  Google Scholar 

  10. Zhao QF, Yu JT, Tan L (2015) S-Nitrosylation in Alzheimer’s disease. Mol Neurobiol 51(1):268–280

    Article  CAS  PubMed  Google Scholar 

  11. Nakamura T, Lipton SA (2010) Preventing Ca2+-mediated nitrosative stress in neurodegenerative diseases: possible pharmacological strategies. Cell Calcium 47(2):90–97

    Article  Google Scholar 

  12. Choi Y-B et al (2000) Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation. Nat Neurosci 3(1):15–21

    Article  CAS  PubMed  Google Scholar 

  13. Forrester MT et al (2009) Detection of protein S-nitrosylation with the biotin-switch technique. Free Radic Biol Med 46(2):119–126

    Article  CAS  PubMed  Google Scholar 

  14. Riederer IM et al (2009) Ubiquitination and cysteine nitrosylation during aging and Alzheimer's disease. Brain Res Bull 80(4-5):233–241

    Article  CAS  PubMed  Google Scholar 

  15. Zahid S et al (2014) Differential S-nitrosylation of proteins in Alzheimer’s disease. Neuroscience 256:126–136

    Article  CAS  PubMed  Google Scholar 

  16. Wang S et al (2012) Two-dimensional nitrosylated protein fingerprinting by using poly (methyl methacrylate) microchips. Lab Chip 12(18):3362–3369

    Article  CAS  PubMed  Google Scholar 

  17. Wang S et al (2011) Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence. J Chromatogr A 1218(38):6756–6762

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zareba-Koziol M et al (2014) Global analysis of S-nitrosylation sites in the wild type (APP) transgenic mouse brain-clues for synaptic pathology. Mol Cell Proteomics 13(9):2288–2305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu C et al (2011) Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach. J Proteome 74(11):2498–2509

    Article  CAS  Google Scholar 

  20. Chen CH et al (2015) Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer’s disease. Neurobiol Dis 76:13–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chouchani ET et al (2010) Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation. Biochem J 430(1):49–59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Forrester MT et al (2009) Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat Biotechnol 27(6):557–559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Su D et al (2013) Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry. Free Radic Biol Med 57:68–78

    Article  CAS  PubMed  Google Scholar 

  24. Pan KT et al (2014) Mass spectrometry-based quantitative proteomics for dissecting multiplexed redox cysteine modifications in nitric oxide-protected cardiomyocyte under hypoxia. Antioxid Redox Signal 20(9):1365–1381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Qu Z et al (2014) Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents. J Proteome Res 13(7):3200–3211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wojdyla K et al (2015) The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations. J Proteome 113:415–434

    Article  CAS  Google Scholar 

  27. Gu L, Robinson RAS (2016) High-throughput endogenous measurement of S-nitrosylation in Alzheimer’s disease using oxidized cysteine-selective cPILOT. Analyst 141(12):3904–3915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Evans AR et al (2015) Global cPILOT analysis of the APP/PS-1 mouse liver proteome. Proteomics Clin Appl 9(9-10):872–884

    Article  CAS  PubMed  Google Scholar 

  29. Evans AR, Robinson RA (2013) Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS(3) acquisition. Proteomics 13(22):3267–3272

    Article  CAS  PubMed  Google Scholar 

  30. Gu L, Evans AR, Robinson RA (2015) Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT. J Am Soc Mass Spectrom 26(4):615–630

    Article  CAS  PubMed  Google Scholar 

  31. Gu L, Robinson RA (2016) A simple isotopic labeling method to study cysteine oxidation in Alzheimer’s disease: oxidized cysteine-selective dimethylation (OxcysDML). Anal Bioanal Chem 408(11):2993–3004

    Article  CAS  PubMed  Google Scholar 

  32. Ting L et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260, USA

    Ryan R. Dyer, Liqing Gu & Renã A. S. Robinson

Authors
  1. Ryan R. Dyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Liqing Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Renã A. S. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Renã A. S. Robinson .

Editor information

Editors and Affiliations

  1. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Enrique Santamaría

  2. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Joaquín Fernández-Irigoyen

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Dyer, R.R., Gu, L., Robinson, R.A.S. (2017). S-Nitrosylation in Alzheimer’s Disease Using Oxidized Cysteine-Selective cPILOT. In: Santamaría, E., Fernández-Irigoyen, J. (eds) Current Proteomic Approaches Applied to Brain Function. Neuromethods, vol 127. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7119-0_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7119-0_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7118-3

  • Online ISBN: 978-1-4939-7119-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation