Identification of protein phosphorylation sites within Ser/Thr-rich cluster domains using site-directed mutagenesis and hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry.
We describe a method for the analysis of multi-site phosphorylation in serine/threonine (Ser/Thr)-rich protein sequences. Site-specific mutagenesis was used to introduce tryptic cleavage sites in the serine glutamine/threonine glutamine cluster domain (SCD) of the human checkpoint protein kinase (Chk2). The mutant proteins were shown to autophosphorylate on residues that are inducibly phosphorylated when mammalian cells are exposed to ionizing radiation (serine 33/35, serine 516, threonine 68 and threonine 432). Five Ser/Thr clusters within the SCD were flanked by arginine or lysine residues to produce tryptic peptides for nanospray liquid chromatography (nanoLC)/linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry. Phosphorylation sites were assigned using accurate-mass-driven analysis and interpretation of low-energy collision-induced dissociation spectra acquired in the ion trap. In addition to verifying known phosphorylation sites, seventeen novel sites were identified within the SCD of Chk2. The approach should be applicable to other O-linked post-translational modifications that occur in proteins with Ser/Thr-rich sequences.
King
Julie B
JB
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Gross
Julia
J
Lovly
Christine M
CM
Piwnica-Worms
Helen
H
Townsend
R Reid
RR
D000595:Amino Acid Sequence; D064447:Checkpoint Kinase 2; D002853:Chromatography, Liquid / Q000379:methods; D017357:Cyclotrons; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D036103:Nanotechnology / Q000379:methods; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013912:Threonine / Q000737:chemistry*
D005973:Glutamine; D013912:Threonine; D012694:Serine; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases