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Profiling Lysine Ubiquitination by Selective Enrichment of Ubiquitin Remnant-Containing Peptides

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1174)

Abstract

Protein ubiquitination plays critical roles in many biological processes. However, functional studies of protein ubiquitination in eukaryotic cells are limited by the ability to identify protein ubiquitination sites. Unbiased high-throughput screening methods are necessary to discover novel ubiquitination sites that play important roles in cellular regulation. Here, we describe an immunopurification approach that enriches ubiquitin remnant-containing peptides to facilitate downstream mass spectrometry (MS) identification of lysine ubiquitination sites. This approach can be utilized to identify ubiquitination sites from proteins in a complex mixture.

Key words

Ubiquitination Ubiquitin remnant-containing peptides Anti-diglycyl lysine antibody Ubiquitin remnant profiling Mass spectrometry Proteomics 

Notes

Acknowledgements

The work was supported by the National Natural Science Foundation of China (Grant 31270874), Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases (BM2013003), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions (GX), NIH-NIMH (MH086128) (SRJ), Boehringer Ingelheim Fonds predoctoral fellowship (AD).

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical SciencesSoochow UniversitySuzhouChina
  2. 2.Department of Pharmacology, Weill Medical CollegeCornell UniversityNew YorkUSA

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