Premium
Sortase A mediated site‐specific immobilization for identification of protein interactions in affinity purification–mass spectrometry experiments
Author(s) -
Kuropka Benno,
Royla Nadine,
Freund Christian,
Krause Eberhard
Publication year - 2015
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201400395
Subject(s) - stable isotope labeling by amino acids in cell culture , chemistry , mass spectrometry , proteomics , linker , tandem affinity purification , affinity chromatography , sortase , computational biology , quantitative proteomics , protein–protein interaction , combinatorial chemistry , biochemistry , chromatography , biology , bacterial protein , computer science , enzyme , gene , operating system
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.