Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches

Analytical Biochemistry
Laetitia FouillenSarah Sanglier-Cianférani

Abstract

The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the ...Continue Reading

References

May 10, 2000·The Journal of Biological Chemistry·D BussoJ M Egly
Oct 26, 2001·Protein Expression and Purification·A M LawrieL N Johnson
Mar 5, 2002·Nature Biotechnology·Scott B FicarroForest M White
Jul 26, 2002·Oncogene·Eberhard SchneiderMathias Montenarh
Oct 11, 2003·Trends in Genetics : TIG·Mario Zurita, Carlos Merino
Nov 9, 2004·Structure·Graziano LolliLouise N Johnson
May 7, 2005·Nature Biotechnology·Thomas A KostDonald L Jarvis
May 26, 2006·Nature Reviews. Molecular Cell Biology·Ole N Jensen
Mar 16, 2007·Cell Cycle·Tarek AbbasAnindya Dutta
Dec 9, 2008·Analytical Biochemistry·Wassim AbdulrahmanArnaud Poterszman
Feb 24, 2009·Protein Expression and Purification·Juan Casado-VelaJ Ignacio Casal

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Citations

Feb 6, 2013·Proceedings of the National Academy of Sciences of the United States of America·Wassim AbdulrahmanArnaud Poterszman

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