Analyzing the Posttranslational Modification Status of Notch Using Mass Spectrometry

  • Shinako Kakuda
  • Robert S. HaltiwangerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)


Notch is modified by multiple types of posttranslational modifications, most of which are known to affect Notch function. The extracellular domain (ECD) is modified with N-glycosylation and at least three types of O-glycosylation (O-fucose, O-glucose, and O-GlcNAc), while the intracellular domain is hydroxylated, phosphorylated, and ubiquitinated. In order to analyze the structure and function of the O-glycans decorating the ECD, we have developed semiquantitative mass spectral methods for identifying modifications at individual sites on Notch that are generally applicable to most posttranslational modifications. Here we describe the expression and purification of Notch ECD fragments, digestion of the fragments with proteases to prepare for mass spectral analysis, and identification of peptides modified with O-glycans using mass spectrometry.

Key words

Notch EGF repeats O-fucose O-glucose O-GlcNAc Mass spectral analysis 



We would like to thank Haltiwanger lab members for helpful comments. Primary work introduced here was supported by NIH grants GM061126 and CA12307101.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Cell BiologyStony Brook UniversityStony BrookUSA

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