Sortase A mediated site-specific immobilization for identification of protein interactions in affinity purification-mass spectrometry experiments

Proteomics. 2015 Apr;15(7):1230-4. doi: 10.1002/pmic.201400395. Epub 2015 Feb 3.

Abstract

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein-protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C-terminal linker region. As a result of this site-directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC-based pull-down experiments and demonstrate the suitability of the approach.

Keywords: Mass spectrometry; Protein interactions; SILAC; Sortase A; Technology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aminoacyltransferases / chemistry*
  • Bacterial Proteins / chemistry*
  • Chromatography, Affinity
  • Cysteine Endopeptidases / chemistry*
  • Enzymes, Immobilized / chemistry*
  • Humans
  • Jurkat Cells
  • Protein Interaction Mapping / methods*
  • Proteomics
  • Tandem Mass Spectrometry

Substances

  • Bacterial Proteins
  • Enzymes, Immobilized
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases