Discovery of Cell Compartment Specific Protein鈥揚rotein Interactions using Affinity Purification Combined with Tandem Mass Spectrometry
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- 作者：Mathieu Lavall茅e-Adam ; Justine Rousseau ; C茅line Domecq ; Annie Bouchard ; Diane Forget ; Denis Faubert ; Mathieu Blanchette ; Benoit Coulombe
- 刊名：Journal of Proteome Research
- 出版年：2013
- 出版时间：January 4, 2013
- 年：2013
- 卷：12
- 期：1
- 页码：272-281
- 全文大小：453K
- 年卷期：v.12,no.1(January 4, 2013)
- ISSN：1535-3907
文摘
Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A.