Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry.
A microelectrospray ionization tandem Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS(n)) approach for structural characterization of protein phosphorylation is described. Identification of proteolytic peptides is based solely upon mass measurement by high field (9.4 Tesla) FT-ICR MS. The location of the modification within any phosphopeptide is then established by FT-ICR MS(2) and MS(3) experiments. Structural information is maximized by use of electron capture dissociation (ECD) and/or infrared multiphoton dissociation (IRMPD). The analytical utility of the method is demonstrated by characterization of protein kinase A (PKA) phosphorylation. In a single FT-ICR MS experiment, 30 PKA tryptic peptides (including three phosphopeptides) were mass measured by internal calibration to within an absolute mean error of |0.7 ppm|. The location of each of the three sites of phosphorylation was then determined by MS(2) and MS(3) experiments, in which ECD and IRMPD provide complementary peptide sequence information. In two out of three cases, electron irradiation of a phosphopeptide [M + nH](n+) ion produced an abundant charge-reduced [M + nH]((n-1)+*) ion, but few sequence-specific c and z(*) fragment ions. Subsequent IRMPD (MS(3)) of the charge-reduced radical ion resulted in the detection of a large number of ECD-type ion products (c and z ions), but no b or y type ions. The utility of activated ion ECD for the characterization of tryptic phosphopeptides was then demonstrated.
Chalmers
Michael J
MJ
National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310-4005, USA.
Håkansson
Kristina
K
Johnson
Robert
R
Smith
Richard
R
Shen
Jianwei
J
Emmett
Mark R
MR
Marshall
Alan G
AG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D017357:Cyclotrons; D005583:Fourier Analysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin