Specificity of MOAC enrichment applied for mature pollen phosphoproteomics studies

0387762 - ÚEB 2013 RIV US eng C - Konferenční příspěvek (zahraniční konf.)
Fíla, Jan - Matros, A. - Čapková, Věra - Mock, H. P. - Honys, David
Specificity of MOAC enrichment applied for mature pollen phosphoproteomics studies.
Proceeding of International Conference on Biomedical Engineering and Biotechnology. Washington, D.C: IEEE Computer Society, 2012 - (Sloan, K.), s. 523-526. ISBN 978-0-7695-4706-0.
[International Conference on Biomedical Engineering and Biotechnology /2012/. Macau (CN), 28.05.2012-30.05.2012]
Grant CEP: GA ČR(CZ) GAP501/11/1462; GA MŠMT OC08011
Výzkumný záměr: CEZ:AV0Z50380511
Klíčová slova: androgenesis * male gametophyte * phosphoproteins
Kód oboru RIV: ED - Fyziologie
http://home.ueb.cas.cz/publikace/2012_Fila_IEEE.pdf

Androgenesis represents one of the most important and fast-developing biotechnological approaches for the production of haploid and di-haploid crop plants with great potential for plant breeding. Tobacco serves as a suitable model plant with available protocols for androgenesis. However, it is necessary to understand the molecular regulatory mechanisms underlying this process. Phosphorylation is the most dynamic posttranslational modification. In a time, the cell contains only few percent of phosphoproteins that can furthermore be accompanied by their native forms. Thus, the enrichment techniques have to be applied in order to concentrate the phosphorylated forms. The aim of our studies was to compare the phosphoproteins extracted by TCA/acetone out of mature pollen and 30-min-activated pollen. The crude protein extracts were enriched by the metaloxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix. The efficiency of the enrichment was verified by 1D SDS-PAGE of the fractions coming out of the enrichment. The separated phosphoproteins were detected by phospho-specific ProQ Diamond stain (Invitrogen) whilst total proteins were stained by colloidal CBB G250. In this short paper, we present our aims in revealing the MOAC specificity for phosphoprotein enrichment. The enrichment of dephosphorylated mature pollen crude extract and the original mature pollen crude extract were compared. Since alkaline phosphatase did not accomplish the dephosphorylation step properly, we decided to apply cerium dioxide for sample dephosphorylation. Since cerium dioxide captured the phosphoproteins rather than dephosphorylated them, the proof of MOAC specificity was impossible to be achieved by these experiments.
Trvalý link: http://hdl.handle.net/11104/0216707