Proteomic Analysis of SUMOylation in the Post-ischemic Brain

  • J. Will Thompson
  • Meng Jiang
  • Wei Yang
Part of the Neuromethods book series (NM, volume 127)


Post-translational protein modification by small ubiquitin-like modifier (SUMO) is increasingly recognized as a key modulator in many cellular processes including DNA repair, cell-cycle regulation, gene transcription, RNA processing, and protein quality control. This modification (SUMOylation) has been implicated in a variety of human diseases of major clinical significance. For example, brain ischemia/reperfusion dramatically activates global protein SUMOylation, which is believed to protect the brain from ischemic injury. Thus, identifying the proteins that are SUMOylated in the post-ischemic brain will provide insight into this endogenous neuroprotective response and may inform the development of new therapeutic strategies. Recent advancement in SUMO proteomics has established reliable methods for systematic characterization of SUMO targets in cells. However, identification of disease-related SUMOylated proteins in complex tissue samples is still technically challenging. Here, we provide a detailed protocol that uses SUMO transgenic mice to characterize SUMOylated proteins in post-ischemic brain samples. We describe the approach and procedures for nuclear fraction preparation, affinity purification of SUMOylated proteins, mass spectrometric data collection, data analysis, and verification of bona fide SUMO targets. This method may be adapted for any tissue sample for analysis of the SUMOylated proteome related to diseases that can be modeled in SUMO transgenic mice.

Key words

SUMO Brain ischemia Stroke Proteomics Transgenic mouse Neuroprotection Tissue Disease 



We thank Kathy Gage for her excellent editorial contributions. This research was supported by AHA Scientist Development Grant 12SDG11950003 and NIH R01 grants NS099590 and NS081299.


  1. 1.
    Flotho A, Melchior F (2013) Sumoylation: a regulatory protein modification in health and disease. Annu Rev Biochem 82:357–385CrossRefPubMedGoogle Scholar
  2. 2.
    Yang W, Paschen W (2015) SUMO proteomics to decipher the SUMO-modified proteome regulated by various diseases. Proteomics 15(5-6):1181–1191CrossRefPubMedGoogle Scholar
  3. 3.
    Nacerddine K et al (2005) The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice. Dev Cell 9(6):769–779CrossRefPubMedGoogle Scholar
  4. 4.
    Yuan H et al (2010) Small ubiquitin-related modifier paralogs are indispensable but functionally redundant during early development of zebrafish. Cell Res 20(2):185–196CrossRefPubMedGoogle Scholar
  5. 5.
    Wang L et al (2014) SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep 15(8):878–885CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hendriks IA, Vertegaal AC (2016) A comprehensive compilation of SUMO proteomics. Nat Rev Mol Cell Biol 17(9):581–595CrossRefPubMedGoogle Scholar
  7. 7.
    Hendriks IA, Vertegaal AC (2016) A high-yield double-purification proteomics strategy for the identification of SUMO sites. Nat Protoc 11(9):1630–1649CrossRefPubMedGoogle Scholar
  8. 8.
    Tammsalu T et al (2015) Proteome-wide identification of SUMO modification sites by mass spectrometry. Nat Protoc 10(9):1374–1388CrossRefPubMedGoogle Scholar
  9. 9.
    Tammsalu T et al (2014) Proteome-wide identification of SUMO2 modification sites. Sci Signal 7(323):rs2CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hendriks IA et al (2014) Uncovering global SUMOylation signaling networks in a site-specific manner. Nat Struct Mol Biol 21(10):927–936CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Tirard M et al (2012) In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice. Proc Natl Acad Sci U S A 109(51):21122–21127CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Yang W et al (2014) Small ubiquitin-like modifier 3-modified proteome regulated by brain ischemia in novel small ubiquitin-like modifier transgenic mice: putative protective proteins/pathways. Stroke 45(4):1115–1122CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yang W et al (2008) Transient focal cerebral ischemia induces a dramatic activation of small ubiquitin-like modifier conjugation. J Cereb Blood Flow Metab 28(5):892–896CrossRefPubMedGoogle Scholar
  14. 14.
    Yang W et al (2008) Transient global cerebral ischemia induces a massive increase in protein sumoylation. J Cereb Blood Flow Metab 28(2):269–279CrossRefPubMedGoogle Scholar
  15. 15.
    Wilm M et al (1996) Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379(6564):466–469CrossRefPubMedGoogle Scholar
  16. 16.
    Zhou W et al (2012) The spectra count label-free quantitation in cancer proteomics. Cancer Genomics Proteomics 9(3):135–142PubMedPubMedCentralGoogle Scholar
  17. 17.
    Schilling B et al (2012) Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation. Mol Cell Proteomics 11(5):202–214CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Rossner MJ, Tirard M (2014) Thy1.2 driven expression of transgenic His(6)-SUMO2 in the brain of mice alters a restricted set of genes. Brain Res 1575:1–11CrossRefPubMedGoogle Scholar
  19. 19.
    Tirard M, Brose N (2016) Systematic localization and identification of SUMOylation substrates in knock-in mice expressing affinity-tagged SUMO1. Methods Mol Biol 1475:291–301CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Proteomics and Metabolomics Shared Resource, Center for Genomic and Computational Biology, Department of Pharmacology and Cancer BiologyDuke University Medical CenterDurhamUSA
  2. 2.Multidisciplinary Neuroprotection Laboratories, Department of AnesthesiologyDuke University Medical CenterDurhamUSA
  3. 3.Department of AnesthesiologyRenmin Hospital of Wuhan UniversityWuhanChina

Personalised recommendations