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

Protocol
Part of the Neuromethods book series (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.

Key words

Cysteine Redox proteomics S-Nitrosylation Alzheimer’s disease Multiplexing 

References

  1. 1.
    Butterfield DA et al (2014) Mass spectrometry and redox proteomics: applications in disease. Mass Spectrom Rev 33(4):277–301CrossRefPubMedGoogle Scholar
  2. 2.
    Lennicke C et al (2016) Redox proteomics: Methods for the identification and enrichment of redox-modified proteins and their applications. Proteomics 16(2):197–213CrossRefPubMedGoogle Scholar
  3. 3.
    Foster MW, Hess DT, Stamler JS (2009) Protein S-nitrosylation in health and disease: a current perspective. Trends Mol Med 15(9):391–404CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Verrastro I et al (2015) Mass spectrometry-based methods for identifying oxidized proteins in disease: advances and challenges. Biomol Ther 5(2):378–411Google Scholar
  5. 5.
    Couvertier SM, Zhou Y, Weerapana E (2014) Chemical-proteomic strategies to investigate cysteine posttranslational modifications. Biochim Biophys Acta 1844(12):2315–2330CrossRefPubMedGoogle Scholar
  6. 6.
    García-Santamarina S, Boronat S, Hidalgo E (2014) Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction. Biochemistry 53(16):2560–2580CrossRefPubMedGoogle Scholar
  7. 7.
    Gu L, Robinson RA (2016) Proteomic approaches to quantify cysteine reversible modifications in aging and neurodegenerative diseases. Proteomics Clin Appl 10(12):1159–1177CrossRefPubMedGoogle Scholar
  8. 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):6160–16169CrossRefGoogle Scholar
  9. 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):7–20CrossRefPubMedGoogle Scholar
  10. 10.
    Zhao QF, Yu JT, Tan L (2015) S-Nitrosylation in Alzheimer’s disease. Mol Neurobiol 51(1):268–280CrossRefPubMedGoogle Scholar
  11. 11.
    Nakamura T, Lipton SA (2010) Preventing Ca2+-mediated nitrosative stress in neurodegenerative diseases: possible pharmacological strategies. Cell Calcium 47(2):90–97CrossRefGoogle Scholar
  12. 12.
    Choi Y-B et al (2000) Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation. Nat Neurosci 3(1):15–21CrossRefPubMedGoogle Scholar
  13. 13.
    Forrester MT et al (2009) Detection of protein S-nitrosylation with the biotin-switch technique. Free Radic Biol Med 46(2):119–126CrossRefPubMedGoogle Scholar
  14. 14.
    Riederer IM et al (2009) Ubiquitination and cysteine nitrosylation during aging and Alzheimer's disease. Brain Res Bull 80(4-5):233–241CrossRefPubMedGoogle Scholar
  15. 15.
    Zahid S et al (2014) Differential S-nitrosylation of proteins in Alzheimer’s disease. Neuroscience 256:126–136CrossRefPubMedGoogle Scholar
  16. 16.
    Wang S et al (2012) Two-dimensional nitrosylated protein fingerprinting by using poly (methyl methacrylate) microchips. Lab Chip 12(18):3362–3369CrossRefPubMedGoogle Scholar
  17. 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):6756–6762CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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):2288–2305CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wu C et al (2011) Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach. J Proteome 74(11):2498–2509CrossRefGoogle Scholar
  20. 20.
    Chen CH et al (2015) Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer’s disease. Neurobiol Dis 76:13–23CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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):49–59CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Forrester MT et al (2009) Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat Biotechnol 27(6):557–559CrossRefPubMedPubMedCentralGoogle Scholar
  23. 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 57:68–78CrossRefPubMedGoogle Scholar
  24. 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):1365–1381CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Qu Z et al (2014) Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents. J Proteome Res 13(7):3200–3211CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wojdyla K et al (2015) The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations. J Proteome 113:415–434CrossRefGoogle Scholar
  27. 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):3904–3915CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Evans AR et al (2015) Global cPILOT analysis of the APP/PS-1 mouse liver proteome. Proteomics Clin Appl 9(9-10):872–884CrossRefPubMedGoogle Scholar
  29. 29.
    Evans AR, Robinson RA (2013) Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS(3) acquisition. Proteomics 13(22):3267–3272CrossRefPubMedGoogle Scholar
  30. 30.
    Gu L, Evans AR, Robinson RA (2015) Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT. J Am Soc Mass Spectrom 26(4):615–630CrossRefPubMedGoogle Scholar
  31. 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):2993–3004CrossRefPubMedGoogle Scholar
  32. 32.
    Ting L et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

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

Personalised recommendations