title: Revealing phosphoproteins playing role in tobacco pollen activated in vitro.
abstract: 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.
authors.affiliation: Laboratory of Pollen Biology, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Fíla
authors.forename: Andrea
authors.initials: A
authors.lastname: Matros
authors.forename: Sonja
authors.initials: S
authors.lastname: Radau
authors.forename: René Peiman
authors.initials: RP
authors.lastname: Zahedi
authors.forename: Věra
authors.initials: V
authors.lastname: Capková
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Mock
authors.forename: David
authors.initials: D
authors.lastname: Honys
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D011058:Pollen / Q000737:chemistry*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D016415:Sequence Alignment; D014025:Titanium; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; D010940:Plant Proteins; D020543:Proteome; C009495:titanium dioxide; D014025:Titanium