A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis.

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.

Cell cycle-dependent phosphorylation of MAN1.

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.