Baessmann, C.

Colby, T.

Hufnagel, P.

Lubeck, M.

Röhrig, H.

Schmidt, J.

Suckau, D.

English

MPG.PuRe

29.8Identification of New in VivoPhosphorylation Sites of EBV-LMP1 byMass SpectrometryL. Chi1, K. Chien2, Y. Chang3, J. Yu4, and L. Fan51Chang Gung University, Kwei-sha, Tao-yuan, Taiwan; 2Departmentof Biochemistry, Graduate Institute of Basic Medical Sciences,Chang Gung University, Kwei-sha, Tao-yuan, Taiwan; 3GraduateInstitute of Basic Medical Sciences, Chang Gung University,Kwei-sha, Tao-yuan, Taiwan; 4Department of Cell and MolecularBiology, Graduate Institute of Basic Medical Sciences, Chang GungUniversity, Kwei-sha, Tao-yuan, Taiwan; 5Department of MedicalTechnology, Yuan-pei Institute of Science and Technology,Hsinchu, TaiwanEpstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is thekey oncoprotein in EBV-associated carcinoma. The LMP1 protein mimicscellular receptor to modulate plenty cellular signal transductions throughself-aggregation and phosphorylation. The phosphorylation of LMP1 canbe detected in EBV-infected or LMP1-gene transfected cell lines. Twophosphorylation sites, S313 and T324, have been predicted by site-directed mutagenesis. However there are additional phosphorylation sitesthat may involve in regulating the turnover rate of LMP1 protein. By in vitrokinase assay, we have demonstrated that the protein casein kinase 2(CK2) is a potent LMP1 kinase. For further understanding whether thisphosphorylation can be detected in vivo, the LMP1 protein was immuno-precipitated from flag-LMP1 gene transfected HEK293T cell lysate andcharacterized by using MALDI-TOF/TOF and LC-ESI MS/MS spectrome-try. We found that there are one to two phosphorylation residues locatedin the carboxyl-terminus activation domain 1 (CTAR1) of LMP1. S211 andS215 are the two candidate amino acids. Since S211 is located in con-sensus CK2 recognition sites, whether LMP1 can be phosphorylated byCK2 in vivo will be further studied.29.9A Strategy for Phosphoproteomics ThatIntegrates Various Gel- and MS-basedTechniquesT. Colby1, P. Hufnagel2, M. Lubeck2, D. Suckau2,C. Baessmann2, J. Schmidt1, and H. Roehrig31Max-Planck-Institut Für Züchtungsforschung, Köln, Germany;2Bruker Daltonik Gmbh, Bremen, Germany; 3University of Bonn,Bonn, GermanyObjective: Protein phosphorylations have an outstanding role in signaltransduction pathways and are thus of significant importance in disease-related studies. Therefore, reliable methods are needed for the detectionand characterisation of phosphorylation sites in large numbers of proteins.Because of its well characterised genome and the relatively high abun-dance of phosphorylations, Arabidopsis thaliana is a convenient modelorganism. So far, many phosphorylation studies deal with individual pro-teins that are purified in order to submit them to extensive analysisapproaches. Lately, a commercial staining kit became available that allowsto selectively stain phosphoproteins in a 2D gel. This allows to parallelisephosphoprotein analysis and thus identify a high number of phosphoryl-ated proteins.Methods: After homogenisation, leaves of A. thaliana were submitted to2D-gel-electrophoresis. For phosphoprotein analysis, the gel was stainedusing the Pro-Q-Diamond-stain (MolecularProbes). After a comparisonwith a total-protein stain, spots of various intensities were picked anddigested. After PMF identification (MALDI-TOF-MS), aliquots of the po-tentially phosphorylated spots were submitted to an in-depth LC-MS/MSanalysis.Results and Conclusion: When a gel of Arabidopsis leaf material isstained using the phosphoprotein-specific dye, surprisingly many spotsappear. Part of them, by comparison to the total-protein image of aSypro-Ruby-stained gel and also MS identification results, seem to befalse positives. The application of a phospho-sensitive stain allows astepwise approach: Candidates that do not seem phosphorylated areeliminated in the staining and/or the MS identification step. Thus, theworkload for LC-MS/MS analytics is reduced. This strategy can be appliednot only to plants but to various kinds of (sub-)proteomes.HUPO 4th Annual World Congress, August 28–September 1, 2005, MunichS276 Molecular & Cellular Proteomics 4.8 by on February 7, 2007 www.mcponline.orgDownloaded from 

A strategy for phosphoproteomics that integrates various gel- and MS-based techniques

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A strategy for phosphoproteomics that integrates various gel- and MS-based techniques

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