Affinity purification strategies for proteomic analysis of transcription factor complexes.
Affinity purification (AP) coupled to mass spectrometry (MS) has been successful in elucidating protein molecular networks of mammalian cells. These approaches have dramatically increased the knowledge of the interconnectivity present among proteins and highlighted biological functions within different protein complexes. Despite significant technical improvements reached in the past years, it is still challenging to identify the interaction networks and the subsequent associated functions of nuclear proteins such as transcription factors (TFs). A straightforward and robust methodology is therefore required to obtain unbiased and reproducible interaction data. Here we present a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding protein alpha (C/EBPalpha). Utilizing the advantages of a double tag and three different MS strategies, we conducted a total of six independent AP-MS strategies to analyze the protein-protein interactions of C/EBPalpha. The resultant data were combined to produce a cohesive C/EBPalpha interactome. Our study describes a new methodology that robustly identifies specific molecular complexes associated with transcription factors. Moreover, it emphasizes the existence of TFs as protein complexes essential for cellular biological functions and not as single, static entities.
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT 25.3, 1090 Vienna, Austria.
Roberto
R
Giambruno
Florian
F
Grebien
Alexey
A
Stukalov
Christian
C
Knoll
Melanie
M
Planyavsky
Elena L
EL
Rudashevskaya
Jacques
J
Colinge
Giulio
G
Superti-Furga
Keiryn L
KL
Bennett
D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity; D056148:Chromatography, Reverse-Phase; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D051381:Rats; D019809:Streptavidin / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D022763:CCAAT-Enhancer-Binding Protein-alpha; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D019809:Streptavidin