RESUMO
For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.
Assuntos
Fosfopeptídeos/isolamento & purificação , Fosfoproteínas/química , Proteômica/métodos , Animais , Automação , Citosol/química , Espectrometria de Massas , Fosfopeptídeos/química , Fosfoproteínas/análise , Proteômica/instrumentação , Reprodutibilidade dos Testes , XenopusRESUMO
The LEM (LAP2beta, Emerin, and MAN1) proteins are essential for nuclear membrane targeting to chromatin via an association with barrier-to-autointegration factor (BAF). Herein, we focused on the mitotic phosphorylation of MAN1 and its biological role. MAN1 was phosphorylated in a cell cycle-dependent manner in the Xenopus egg cell-free system, and the mitotic phosphorylation at the N-terminal region of MAN1 suppressed the binding of MAN1 to BAF. Titansphere column chromatography followed by MS/MS sequencing identified at least three M-phase-specific phosphorylation sites, Thr-209, Ser-351, and Ser-402, and one cell cycle-independent phosphorylation site, Ser-463. An in vitro BAF binding assay involving mutants S402A and S402E suggested that the phosphorylation of Ser-402 was important for regulation of the binding of MAN1 to BAF.
