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


Simicevic, Jovan; Schmid, Adrien W; Gilardoni, Paola A; Zoller, Benjamin; Raghav, Sunil K; Krier, Irina; Gubelmann, Carinne; Lisacek, Frédérique; Naef, Felix; Moniatte, Marc; Deplancke, Bart (2013). Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics. Nature Methods, 10(6):570-576.

Abstract

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 PPARɣ 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 DNnA binding model for PPARɣ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARɣ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARɣ copy number.

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 PPARɣ 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 DNnA binding model for PPARɣ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARɣ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARɣ copy number.

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33 citations in Web of Science®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:Special Collections > SystemsX.ch
Special Collections > SystemsX.ch > Research, Technology and Development Projects > CycliX
Special Collections > SystemsX.ch > Research, Technology and Development Projects
Special Collections > SystemsX.ch > Interdisciplinary PhD Projects
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2013
Deposited On:15 Jul 2013 13:07
Last Modified:05 Apr 2016 16:50
Publisher:Nature Publishing Group
ISSN:1548-7091
Publisher DOI:https://doi.org/10.1038/nmeth.2441
PubMed ID:23584187
Permanent URL: https://doi.org/10.5167/uzh-78970

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