An Integrated Strategy for Identifying Targets of Ubiquitin-Mediated Degradation in CD4+ T Cells

  • Natania S. Field
  • Claire E. O’Leary
  • Joseph M. Dybas
  • Hua Ding
  • Paula M. OliverEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2111)


Ubiquitination is a crucial component of many immune processes. While ubiquitin-mediated degradation is essential to T cell activation via T cell receptor signaling, the specific E3 ligases and substrates involved are not well-understood. Here, we describe a strategy integrating RNA, protein, and posttranslational modification datasets to identify targets of ubiquitin-mediated degradation. When integrated, these assays can provide broad insight into how this posttranslational modification regulates protein function and influences T cell biology.

Key words

Ubiquitin T cell Lymphocyte Diglycine remnant profiling Signaling 


  1. 1.
    Fang D et al (2002) Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat Immunol 3:281–287CrossRefGoogle Scholar
  2. 2.
    Layman AAK et al (2017) Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease. Nat Commun.
  3. 3.
    Layman AAK, Oliver PM (2016) Ubiquitin ligases and deubiquitinating enzymes in CD4+ T cell effector fate choice and function. J Immunol 196:3975–3982CrossRefGoogle Scholar
  4. 4.
    Dybas JM et al (2019) Integrative proteomics reveals that CD4+ T cell activation promotes predominantly non-degradative ubiquitylation. Nat Immunol 20(6):747–755CrossRefGoogle Scholar
  5. 5.
    Udeshi ND et al (2013) Refined preparation and use of anti-diglycine remnant (K-e-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments. Mol Cell Proteomics 132:825–831CrossRefGoogle Scholar
  6. 6.
    Mertins P et al (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10:634–637CrossRefGoogle Scholar
  7. 7.
    Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. Scholar
  8. 8.
    Love MI, Huber W, Anders S (2011) Targeted analysis of nucleotide and copy number variation by exon capture in allotetraploid wheat genome. Genome Biol 15:550CrossRefGoogle Scholar
  9. 9.
    Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. ISBN 978-3-319-24277-4Google Scholar
  10. 10.
    Dobin A et al (2013) Sequence analysis STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21CrossRefGoogle Scholar
  11. 11.
    Dyballa N, Metzger S (2009) Fast and sensitive colloidal Coomassie G-250 staining for proteins in polyacrylamide gels. Part 1: Two-dimensional (2-D) gel electrophoresis using cup-loading Part 2: Colloidal Coomassie staining with CBB G-250. J Vis Exp.
  12. 12.
    Baruzzo G et al (2017) Simulation-based comprehensive benchmarking of RNA-seq aligners HHS Public Access. Nat Methods 14:135–139CrossRefGoogle Scholar
  13. 13.
    Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319CrossRefGoogle Scholar
  14. 14.
    Geer LY et al (2004) Open mass spectrometry search algorithm, J Proteome Res. Scholar
  15. 15.
    Tan H et al (2017) Integrative proteomics and phosphoproteomics profiling reveals dynamic signaling networks and bioenergetics pathways underlying T cell activation. Immunity 46:488–503CrossRefGoogle Scholar
  16. 16.
    Pascovici D, Handler DCL, Wu JX, Haynes PA (2016) Multiple testing corrections in quantitative proteomics: a useful but blunt tool. Proteomics 16:2448–2453. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Natania S. Field
    • 1
    • 2
  • Claire E. O’Leary
    • 4
  • Joseph M. Dybas
    • 1
    • 2
  • Hua Ding
    • 3
  • Paula M. Oliver
    • 1
    • 2
    Email author
  1. 1.The University of PennsylvaniaPhiladelphiaUSA
  2. 2.Division of Protective ImmunityDepartment of Pathology and Laboratory Medicine, The Children‘s Hospital of PhiladelphiaPhiladelphiaUSA
  3. 3.Cell Pathology DivisionDepartment of Pathology and Laboratory Medicine, The Children‘s Hospital of PhiladelphiaPhiladelphiaUSA
  4. 4.Department of MedicineUniversity of California-San FranciscoSan FranciscoUSA

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