An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.
Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.
Bürckstümmer
Tilmann
T
Research Center for Molecular Medicine (CeMM), Lazarettgasse 19/3, 1090 Vienna, Austria.
Bennett
Keiryn L
KL
Preradovic
Adrijana
A
Schütze
Gregor
G
Hantschel
Oliver
O
Superti-Furga
Giulio
G
Bauch
Angela
A
D000345:Affinity Labels / Q000302:isolation & purification / Q000378:metabolism; D002468:Cell Physiological Phenomena; D002846:Chromatography, Affinity / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D020543:Proteome / Q000302:isolation & purification* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
D000345:Affinity Labels; D020543:Proteome