期刊
PROTEOMICS
卷 12, 期 21, 页码 3229-3250出版社
WILEY
DOI: 10.1002/pmic.201100318
关键词
Male gametophyte; Metal oxide affinity chromatography; Nicotiana tabacum; Phosphoproteomics; Plant proteomics; Titanium dioxide (TiO2)
资金
- ESF (COST Action) [0603, COST-STSM-FA0603-04559, COST-STSM-FA0603-05564]
- Czech Science Foundation [P501/11/1462, P305/12/2611]
- German Academic Exchange Service [50755371]
- DAAD/ASCR bilateral project [D8-CZ19/2011-2012]
- European Fund for Regional Development, the Operational Program Prague-Competitiveness [CZ.2.16/3.1.00/21159]
The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.
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