Mass spec-based counting of transcription factors during adipogenesis
© 2013 EPFL
Nature Methods presents a new method to simultaneously quantify up to 10 TFs by SRM-MS in absolute terms and to model their behavior, developed in collaboration by the group of Prof. Bart Deplancke together with colleagues of Prof. Félix Naef’s lab and the EPFL Proteomics Core Facility, coordinated by Dr. Marc Moniatte.
The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring mass spectrometry–based assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of some key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ~250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.
These interesting results are a perfect example of the benefits of a tight and fruitful collaboration between Prof. Deplancke and Prof. Naef's groups and the EPFL Proteomics Core Facility team, headed by Dr Marc Moniatte.
The Publication in Nature Methods of April 14, 2013: "Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics"