Identification of SUMO E3 ligase-specific substrates using the HuProt human proteome microarray

Methods Mol Biol. 2015;1295:455-63. doi: 10.1007/978-1-4939-2550-6_32.

Abstract

The functional protein microarray is a powerful and versatile systems biology and proteomics tool that allows the rapid activity profiling of thousands of proteins in parallel. We have recently developed a human proteome array, the HuProt array, which includes ~80 % of all the full-length proteins of the human proteome. In one recent application of the HuProt array, we identified numerous SUMO E3 ligase-dependent SUMOylation substrates. For many SUMO E3 ligases, only a small number of substrates have been identified and the target specificities of these ligases therefore remain poorly defined. In this protocol, we outline a method we developed using the HuProt array to screen the human proteome to identify novel SUMO E3 ligase substrates recognized by specific E3 ligases.

MeSH terms

  • Humans
  • Protein Array Analysis / methods*
  • Protein Processing, Post-Translational
  • Proteome*
  • Proteomics / methods*
  • Small Ubiquitin-Related Modifier Proteins / isolation & purification
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Substrate Specificity
  • Sumoylation
  • Ubiquitin-Protein Ligases / isolation & purification
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Proteome
  • Small Ubiquitin-Related Modifier Proteins
  • Ubiquitin-Protein Ligases