Phosphoproteomics

Dr. Martin R. Larsen
Universtiy of Southern Denmark

McIntyre Medical Building, Room 521

Phosphoproteomics

We have developed robust strategies for the specific enrichment of phosphorylated peptides from complex mixtures, including titanium dioxide procedures (1) and procedures for the separation of multiphosphorylated peptides from monophosphorylated peptides (SIMAC)(2). These strategies we have applied to a variety of biological systems including depolarization signaling in nerve endings.

During the last few years we have performed comprehensive mapping of phosphorylation sites in proteins involved in synaptic vesicle endocytosis (SVE) which is the mechanism with which synaptic vesicles are recruited back into the nerve end after release of neurotransmitters following depolarization of nerve endings. The biological events exocytosis and endocytosis are extremely important and crucial for maintenance of synaptic transmission between nerve cells.

New  strategies has recently been developed for large scale phosphoproteomics aiming at downscaling and optimizing the procedures for very low amount of materials obtainable from primary cells (µg level).  Preliminary data obtained using one of the new strategies on isolated synaptosomes has revealed a large number of peptides carrying multiply phosphorylation sites as well as singly phosphorylation sites, which are only detected with this novel technique. We have presently purified synaptosomes from rat brains and validated the function and quality of these with respect to responses to 10 sec depolarization using 32P-ATP incorporation. We have presently identified >2000 unique phosphorylation sites from very small amount of proteins from synaptosomes using only the new strategy.