Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

作者：Simicevic Jovan; Schmid Adrien W; Gilardoni Paola A; Zoller Benjamin; Raghav Sunil K; Krier Irina; Gubelmann Carine; Lisacek Frederique; Naef Felix; Moniatte Marc^*; Deplancke Bart

来源：Nature Methods, 2013, 10(6): 570-+.

DOI：10.1038/NMETH.2441

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-based mass spectrometry 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 key TFs, including PPAR gamma and RXR alpha, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from similar to 250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DND binding model for PPAR gamma based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPAR gamma binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPAR gamma copy number.

• 出版日期2013-6