Phosphopeptide Mapping

FIGURE 1 Autoradiograms of two-dimensional phosphopeptide maps of LC20 phospho-rylated in aortic homogenate. Six phosphopeptides, labeled A through F, are resolved. From Erdodi et al. (1988b, Hg. 2, p. 159). 

Dephosphorylation of LC20 by myosin light chain phosphatase (MLCP) is described in Chapter 10, this volume. For dephosphorylation of distinct sites of LC20, we refer to Erdodi et al. (1989) and Barany et al. 

Phosphopeptide mapping provides qualitative and quantitative information about the sites phosphory- 

resulting in high resolution (Fig. 1). Six peptides, A-F are shown in Fig. 1, and their characterization is described in Section III.D.2. Based on autoradiography, the powder containing the phosphopeptides can be scraped from the cellulose sheets and counted, and the incorporation of [ PJphosphate into peptides of LC20 can be quantified (Rokolya etal., 

Alternatively, the phosphopeptides can be eluted from the powder and freeze-dried for phospho-amino acid analysis. 

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R.S. Annan, M.J. Huddleston, R. Verma, R.J. Deshaies, S.A. Carr, A multidimensional ESI-MS-based approach to phosphopeptide mapping. Anal. Chem., 73 (2001)393. 

Two-dimensional phosphopeptide maps of LC20 from 32p-iabeled muscles stimulated with different agents allows one to differentiate between the involvement of MLCK and PKC the phosphopeptide map of LC20 isolated from K+-stimulated arteries shows predominantly MLCK/Ser peptides (Erdodi et al, 1987), whereas in phorboldibutyrate (PDBu)-treated muscle... 

FIGURES Autoradiograms of phosphopeptide maps of isolated LC20 phosphorylated by either MLCK or PKC. From Erdodi et al. (1988a, Fig. 4, p. 587). 

Phosphopeptide Mapping and Phosphoamino Acid Anaiysis on Cellulose Thin-Layer Plates... 

Switch on the high-voltage supply. Typically phosphopeptide maps are run for 20-30 min at 1.0 kV on the HTLE 7000. However, with other types of apparatus the voltage needed has to be determined empirically, and will depend on the distance between the electrodes. 

Boyle, W. J., van der Geer, P., and Hunter, T. (1991b) Phosphopeptide mapping and phos-phoamino acid analysis by two-dimensional separation on cellulose thin-layer plates. In Methods in Enzymology (T. Hunter and B. M. Sefton, eds.), Vol. 201, pp. 110-149. Academic Press, San Diego. 

Flora, J. W. Muddiman, D. C. Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a Penning ion trap An energetically favored process. J. Am. Chem. Soc. 2004, 124, 6546-6547. 

McMichael, R.W.J. and Lagarias, J.C., Phosphopeptide mapping of Avena phytochrome phospho-rylated by protein kinases in vitro. Biochemistry, 29, 3872,1990. 

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