Abstract
A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.
Article PDF
Similar content being viewed by others
References
Matsurbara, M.; Yamauchi, E.; Hayashi, N.; Taniguchi, H. MARCKS, a Major Protein Kinase C Substrate, Assumes Nonhelical Conformations Both in Solution and in Complex with Ca2+-Calmodulin. FEBS Lett. 1998, 421, 203–207.
Stumpo, D. J.; Bock, C. B.; Tuttle, J. S.; Blackshear, P. J. MARCKS Deficiency in Mice Leads to Abnormal Brain Development and Perinatal Death. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 944–948.
Arbuzova, A.; Schmitz, A. A.; Vergeres, G. Cross-talk Unfolded: MARCKS Proteins. Biochem. J. 2002, 362, 1–12.
Arbuzova, A.; Murray, D.; McLaughlin, S. MARCKS, Membranes and Calmodulin: Kinetics of Their Interaction. Biochim. Biophys. Acta 1998, 1376, 369–379.
Brooks, G. The Role of 80K/MARCKS, a Specific Substrate of Protein Kinase C, in Cell Growth and Tumor Progression. Pigment Cell Res. 1994, 7, 451–457.
Trifaro, J. M.; Lejen, T.; Rose, S. D.; Pene, T. D.; Barkar, N. D.; Seward, P. Pathways that Control Cortical F-actin Dynamics During Secretion. Neurochem. Res. 2002, 27, 1371–1385.
Graff, J. M.; Young, T. N.; Johnson, J. D.; Blackshear, P. J. Phosphorylation-Regulated Calmodulin Binding to a Prominent Cellular Substrate for Protein Kinase C. J. Biol. Chem. 1989, 264, 21818–21823.
Taniguchi, H.; Manenti, S. Interaction of Myristoylated Alanine-Rich Protein Kinase C Substrate (MARCKS) with Membrane Phospholipids. J. Biol. Chem. 1993, 268, 9960–9963.
Kim, J.; Shishido, T.; Jiang, X.; Aderem, A.; McLaughlin, S. Phosphorylation, High Ionic Strength, and Calmodulin Reverse the Binding of MARCKS to Phospholipid Vesicles. J. Biol. Chem. 1994, 269, 28214–28219.
Arbuzova, A.; Wang, L.; Wang, J.; Hangyas-Mihalyne, G.; Murray, D.; Honig, B.; McLaughlin, S. Membrane Binding of Peptides Containing Both Basic and Aromatic Residues: Experimental Studies with Peptides Corresponding to the Scaffolding Region of Caveolin and the Effector Region of MARCKS. Biochemistry 2000, 39, 10330–10339.
Hartwig, J. H.; Thelen, M.; Rosen, A.; Janmey, P. A.; Nairn, A. C.; Aderen, A. MARCKS is an Actin Filament Crosslinking Protein Regulated by Protein Kinase C and Calcium-calmodulin. Nature 1992, 356, 618–622.
Tapp, H.; Al-Naggar, I. M.; Yarmola, E. G.; Harrison, A.; Shaw, G.; Edison, A. S.; Bubb, M. MARCKS is a Natively Unfolded Protein with an Inaccessible Actin-binding Site. J. Biol. Chem. 2005, 280, 9946–9956.
Arbuzova, A.; Wang, J.; Murray, D.; Jacob, J.; Cafiso, D. S.; McLaughlin, S. Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes and Calmodulin. J. Biol. Chem. 1997, 43, 271677–227177.
McIlroy, B. K.; Walters, J. D.; Blackshear, P. J.; Johnson, J. D. Phosphorylation-dependent Binding of a Synthetic MARCKS Peptide to Calmodulin. J. Biol. Chem. 1991, 266, 4959–4964.
Palmer, R. H.; Schonwaber, D. C.; Rahman, D.; Pappin, D. J. C.; Herget, T.; Parker, P. J. PRK1 Phosphorylates MARCKS at the PKC Sites: Serine 152, Serine 156 and Serine 163. FEBS Letters 1996, 378, 281–285.
Nagumo, H.; Ikenoya, M.; Sakurada, K.; Furuya, K.; Ikuhara, T.; Hirgaka, H.; Sasaki, Y. Rho-associated Kinase Phosphorylates MARCKS in Human Neuronal Cells. Biochem. Biophys. Res. Commun. 2001, 280, 605–609.
Taniguchi, H.; Manenti, S.; Suzuki, M.; Titani, K. Myristoylated Alanine-rich C Kinase Substrate (MARCKS), a Major Protein Kinase C Substrate, is an in vivo Substrate of Proline-directed Protein Kinases: A Mass Spectrometric Analysis of Post-translational Modifications. J. Biol. Chem. 1994, 269, 18299–18302.
Newton, A. C. Protein Kinase C: Structure, Function, and Regulation. J. Biol. Chem. 1995, 270, 28495–28498.
Rosen, A.; Keenan, K. F.; Thelen, M.; Nairn, A. C.; Aderem, A. Activation of Protein Kinase C Results in the Displacement of its Myristoylated, Alanine-Rich Substrate from Punctate Structures in Macrophage Filopodia. J. Exp. Med. 1990, 172, 1211–1215.
Qin, K.; Cafiso, D. S.; Qin, Z. Membrane Structure of the Protein Kinase C and Calmodulin Binding Domain of Myristoylated Alanine-Rich C Kinase Substrate Determined by Site-directed Spin Labeling. Biochemistry 1996, 35, 2917–2925.
Yarmola, E. G.; Edison, A. S.; Lenox, R. H.; Bubb, M. R. Actin Filament Cross-Linking by MARCKS: Characterization of Two Actin-Binding Sites Within the Phosphorylation Site Domain. J. Biol. Chem. 2001, 276, 22351–22358.
Bubb, M. R.; Lenox, R. H.; Edison, A. S. Phosphorylation-Dependent Conformational Changes Induce a Switch in the Actin-binding Function of MARCKS. J. Biol. Chem. 1999, 274, 36472–36478.
Seki, K.; Sheu, F.; Huang, K. Binding of Myristoylated Alanine-Rich Protein Kinase C Substrate to Phosphoinositides Attenuates the Phosphorylation by Protein Kinase C. Arch. Biochem. Biophys. 1996, 326, 193–201.
Yamauchi, E.; Kiyonami, R.; Kanai, M.; Taniguchi, H. The C-terminal Conserved Domain of MARCKS is Phosphorylated in Vivo by Proline-Directed Protein Kinase. J. Biol. Chem. 1998, 273, 4367–4371.
Arness, B.; Manjarrez-Hernandex, A.; Howell, S. A.; Learmonth, M.; Aitken, A. Multisite Phosphorylation of the 80 kDa (MARCKS) Protein Kinase C Substrate in C3H/10T1/2 Fibroblasts. FEBS Lett. 1992, 297, 285–291.
Shi, S. D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Phosphopeptide/phosphoprotein Mapping by Electron Capture Dissociation Mass Spectrometry. Anal. Chem. 2001, 73, 19–22.
Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nature Biotech. 2003, 21, 255–261.
McLachlin, D. T.; Chait, B. T. Analysis of Phosphorylated Proteins and Peptides by Mass Spectrometry. Curr. Opin. Chem. Biol. 2001, 5, 591–602.
Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology; MCC Press: San Diego, 2003.
Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A Primer. Mass Spectrom. Rev. 1998, 17, 1–35.
Marshall, A. G.; Hendrickson, C. L. Fourier Transform Ion Cyclotron Resonance Detection: Principles and Experimental Configurations. Int. J. Mass Spectrom. 2002, 215, 59–75.
Amster, I. J. Fourier Transform Mass Spectrometry. J. Mass Spectrom. 1996, 31, 1325–1337.
Cooper, H. J.; Hakansson, K.; Marshall, A. G. The Role of Electron Capture Dissociation in Biomolecular Analysis. Mass Spectrom. Rev. 2005, 24, 201–222.
Laskin, J.; Futrell, J. H. Collisional Activation of Peptide Ions in FTICR Mass Spectrometry. Mass Spectrom. Rev. 2003, 22, 158–181.
Goeringer, D.; Duckworth, D.; McLuckey, S. Collison-Induced Dissociation in Quadrupole Ion Traps: Application of a Thermal Model to Diatomic Ions. J. Phys. Chem. A 2001, 105, 1882–1889.
Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Electron Capture Dissociation of Multiply Charged Protein Cations: A Nonergodic Process. J. Am. Chem. Soc. 1998, 120, 3265–3266.
Tsybin, Y. O.; Ramstron, M.; Witt, M.; Baykut, G.; Hakansson, P. Peptide and Protein Characterization by High-rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Mass Spectrom. 2004, 39, 719–729.
McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. Electron Capture Dissociation of Gaseous Multiply Charged Ions by Fourier Transform Ion Cyclotron Resonance. J. Am. Soc. Mass Spectrom. 2001, 12, 245–249.
Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at 9.4 Tesla. Rapid Commun. Mass Spectrom. 1996, 10, 1824–1828.
Hakansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Combined Electron Capture and Infrared Multiphoton Dissociation for Multistage MS/MS in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Anal. Chem. 2003, 75, 3256–3262.
Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. External Accumulation of Ions for Enhanced Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, 970–976.
Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. A High Performance Modular Data System for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Rapid Commun. Mass Spectrom. 1996, 10, 1839–1844.
Blakney, G. T.; van der Rest, G.; Johnson, J. R.; Freitas, M. A.; Drader, J. J; Shi, S. D. H.; Hendrickson, C. L.; Kelleher, N. L.; Marshall, A. G. Further Improvements to the MIDAS Data Station for FT-ICR Mass Spectrometry. Proceedings of the 49th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics; Chicago, IL, June 2001.
Burton, R. D.; Matuszak, K. P.; Watson, C. H.; Eyler, J. R. Exact Mass Measurements Using a 7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer in a Good Laboratory Practices Regulated Environment. J. Am. Soc. Mass Spectrom. 1999, 10, 1291–1297.
Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Automated Reduction and Interpretation of High Resolution Electrospray Mass Spectra of Large Molecules. J. Am. Soc. Mass Spectrom. 2000, 11, 320–332.
LeDuc, R. D.; Taylor, G. K.; Kim, Y.; Januszyk, T. E.; Bynum, L. H.; Sola, J. V.; Garavelli, J. S.; Kelleher, N. L. ProSight PTM: An Integrated Environment for Protein Identification and Characterization by Top Down Mass Spectrometry. Nucleic Acids Res. 2004, 32, W340-W345.
Clauser, K. R.; Baker, P. R.; Burlingame, A. L. Role of Accurate Mass Measurement (+/− 10 ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching. Anal. Chem. 1999, 71, 2871–2882.
Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Electron Capture Dissociation of Singly and Multiply Phosphorylated Peptides. Rapid Commun. Mass Spectrom. 2000, 14, 1793–1800.
Kelleher, N. L. Top Down Proteomics. Anal. Chem. 2004, 76, 197A-203A.
Horn, D. M.; Lindh, I.; McLafferty, F. W. Phosphopeptide/Phosphoprotein Mapping by Electron Capture Dissociation Mass Spectrometry. Anal. Chem. 2001, 73, 19–22.
Chalmers, M. J.; Kolch, W.; Emmett, M. R.; Marshall, A. G.; Mischak, H. Identification and Analysis of Phosphopeptides. J. Chromatogr. B 2004, 803, 111–120.
Guan, S.; Marshall, A. G. Stored Waveform Inverse Fourier Transform (SWIFT) Ion Excitation in Trapped Ion Mass Spectrometry Theory and Applications. Int. J. Mass Spectrom. Ion Processes 1996, 158, 5–37.
Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. Tailored Excitation for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Chem. Soc. 1985, 107, 7893–7897.
Pesavento, J. J.; Mizzen, C. A.; Kelleher, N. L. Quantitative Analysis of Modified Proteins and Their Positional Isomers by Tandem Mass Spectrometry: Human Histone H4. Anal. Chem. 2007, 78, 4271–4280.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online September 20, 2007
Rights and permissions
About this article
Cite this article
Woodling, K.A., Eyler, J.R., Tsybin, Y.O. et al. Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein. J. Am. Soc. Spectrom. 18, 2137–2145 (2007). https://doi.org/10.1016/j.jasms.2007.09.010
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2007.09.010