Poster: Late and Moved Abstracts

Abs # 1384: Analysis of Arabidopsis Phosphoproteins by Mass Spectrometry

Presenter:	Harmon, Alice C, harmon@botany.ufl.edu
Authors	Harmon, Alice C (A)   Hegeman, Adrian  (C)   Strachan, Camille N (E)   Stevens, Stanley M (F)   Harms, Amy  (C)   Sussman, Michael R. (C)   Denslow, Nancy D (B)   Harper, Jeffrey F (D)
Affiliations:	(A): Department of Botany, University of Florida (B): Interdisciplinary Center for Biotechnology Research, University of Florida (C): Biotechnology Center, University of Wisconsin (D): Departement of Cell Biology, The Scripps Research Institution (E): Department of Chemistry, University of Florida (F): Department of Medicinal Chemistry, University of Florida

As a step towards characterizing the substrate specificity of CDPKs, the autophosphorylation sites of a CDPK (CPK4) and a CDPK-related kinase (CRK3) were determined by mass spectrometry. These enzymes were expressed in bacteria as fusions with affinity purification tags, purified, autophosphorylated, denatured in urea, and digested with trypsin. CPK4 tryptic peptides were resolved by tandem chromatography on a strong cation exchange and reverse-phase resins. CRK3 peptides were resolved by reverse-phase chromatography alone. Peptides were sequenced using a Micromass Q-TOF2 mass spectrometer, and the data were analyzed by Mascot software. Phosphopeptides were identified by the loss of 98/z amu from the parent ion in the MS/MS spectra. One phosphorylation site in CPK4 and five sites in CRK3 were identified. To identify proteins endogenously phosphorylated in Arabidopsis, leaves were frozen in liquid nitrogen and extracted with Trizol. In the first strategy, protein bands resolved by SDS-PAGE were excised and digested with trypsin. Phosphopeptides were isolated from the peptide mixture by immobilized gallium affinity chromatography, resolved by reverse-phase capillary chromatography and analyzed on Thermo-Finnigan LCQ-ion trap mass spectrometer. In the second strategy, phosphoproteins were isolated from the extract by immunoaffinity chromatography on a resin of immobilized anti-phosphoserine and anti-phosphothreonine, digested with trypsin, resolved by strong cation exchange and reverse-phase chromatography, and analyzed on an LCQ-ion trap mass spectrometer. Using these techniques we have identified >80 putative phosphoproteins. This work was supported by a grant (MCB 0114769) from the National Science Foundation: to ACH, JFH, MRS, EM Hrabak, and JC Cushman.