Chemical proteomics in plant the methodical preview René Lenobel Group of proteomics, Laboratory of Growth Regulators , Olomouc

Chemical proteomics – basic background o Background Incubation of a sorbent modified with specific ligand with a protein lysate. After washing step, retained proteins are released by elution buffer or by denaturation. Collected proteins are analyzed using proteomic methods. o Application Study of proteins with similar specificity Analysis and identification of putative cell targets of immobilized ligands WASH SDS-PAGE „Shotgun“ approach LC- MS/MS Bioinformatic analysis Lysis Incubation Aff. sorbent Ligand Spacer Matrix Elution

Chemical proteomics: Unbiased but not so straightforward approach Ligand design, its synthesis and immobilization on a solid matrix. Preventing of protein degradation, omitting strong detergents. Resolving between specific and nonspecific interactors Strong washing buffers (high salt conc, weak detergents) Using chemical competitors or unspecific control matrices (similar compounds without specific binding to desired proteins) Quantitative proteomic methods (stable isotope labeling, label-free quantitation)

Chemical proteomics with cytokinins Experimental design Wash Elution SDS-PAGE „Shotgun“ approach LC-MS/MS Lysis Incubation Affinity matrix Ligand Spacer Matrix Bioinformatic analysis 6-benzylaminopurine (BAP) Cytokinin-binding protein 1 (CBP-1) Wheat seeds (T. Aestivum)

BAP affinity sorbent synthesis 2(6-aminobuthylimino)-BAP (C2-BAP)NHS-Fast Flow Sepharose 19 atoms, 30 Å BAP

Small preview of our results Competitor NO 5mM ATP 5mM ATP, 0,1mM BAP Database CBP-custom T. Aestivum UniGene EST Competitor NO5 mM ATP 0,1 mM BAPNO5 mM ATP 0,1 mM BAP Peptide number Sequence coverage19,8%15,4%9,4%40%26%17% Mascot score376,6445,2251, Spectral count emPAI---1,581,030,68 Database T. Aestivum UniGene EST NCBInr Green plants Competitor NO5 mM ATP 0,1 mM BAPNO5 mM ATP 0,1 mM BAP Number of ID proteins kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 200 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa 200 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa

Putative targets of BAP S-adenosyl-L-homocystein hydrolase Methionin synthetase S-adenosyl-L-methionine synthase

Acknowledgement LRR – proteomic group Ivo Chamrád Radim Simerský Lucie Švehlová prof. ing. Miroslav Strnad, CSc. LRR – organic synthesis group Lucie Szüčová Václav Mik Jindřich Kania Marek Zatloukal And many other people for fruitfull discussions and advices Thank you for your attention