Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches

• Published in: Analytical biochemistry Vol. 407; no. 1; pp. 34 - 43
• Main Authors: Fouillen, Laetitia, Abdulrahman, Wassim, Moras, Dino, Dorsselaer, Alain Van, Poterszman, Arnaud, Sanglier-Cianférani, Sarah
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-2010; Elsevier Masson
• Subjects: Amino Acid Sequence; Baculovirus; Baculovirus expression system; Biochemistry, Molecular Biology; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; CDK-activating kinase (CAK); Chromatography, High Pressure Liquid - methods; Coexpression; Cyclin H - genetics; Cyclin H - isolation & purification; Cyclin H - metabolism; Cyclin-Dependent Kinase 2 - metabolism; Cyclin-Dependent Kinases - genetics; Cyclin-Dependent Kinases - isolation & purification; Cyclin-Dependent Kinases - metabolism; Escherichia coli; Life Sciences; Mass spectrometry; Molecular Sequence Data; Nanotechnology - methods; Phosphopeptides - analysis; Phosphoproteins; Phosphoproteins - chemistry; Phosphorylation; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RNA Polymerase II - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Baculovirus expression system; Phosphorylation; CDK-activating kinase (CAK); Phosphoproteins; Mass spectrometry; Coexpression
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.07.006

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 analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1.