Characterization of protein kinase A phosphorylation: multi-technique approach to phosphate mapping

• Published in: Analytical biochemistry Vol. 324; no. 2; pp. 204 - 218
• Main Authors: Shen, Jianwei, Smith, Richard A, Stoll, Vincent S, Edalji, Rohinton, Jakob, Clarissa, Walter, Karl, Gramling, Emily, Dorwin, Sally, Bartley, Diane, Gunasekera, Angelo, Yang, Jianguo, Holzman, Thomas, Johnson, Robert W
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-01-2004
• Subjects: Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Cyclic AMP-Dependent Protein Kinases - metabolism; FT-ICR; HPLC-MS; IMAC; LCQ; MALDI; Mass Spectrometry; Peptide Mapping; Phosphorprotein; Phosphorylation; PKA; Q-Tof; FT-ICR; IMAC; Q-Tof; HPLC-MS; MALDI; PKA; LCQ; Mass spectrometry; Phosphorprotein
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.09.016

A multi-technique approach to identification and mapping of phosphorylation on protein kinase A (PKA) is described. X-ray crystallography revealed phosphorylation at T197 and S338 while mass spectrometry (MS) on the intact protein suggested phosphorylation at three sites. Tryptic digestion, followed by MS, confirmed the presence of three phosphates. However, metal affinity treatment of the digest prior to MS revealed the presence of a fourth phosphopeptide. Subsequent analysis of the digests using liquid chromatography (LC) coupled with quadrupole ion trap (QIT) MS confirmed phosphorylation at S10 and S338 and suggested phosphorylation at S139 and T195/197. Unfortunately, identification of pS139 was inconclusive due to low signal intensity and early elution in reversed-phase LC while poor MS/MS data prevented localization of the phosphate to T195 or T197. Phosphopeptide modification with ethanethiol, followed by LC QIT-MS/MS, identified four phosphopeptides in a single experiment. In addition, the fragmentation data provided significantly more sequence information than data obtained from unmodified peptides. Data from this study suggested that PKA was completely phosphorylated at S10, T197, and S338 and partially phosphorylated at S139. These results illustrate that critical information can be lost unless multiple MS techniques are used for identification and validation of phosphorylation.