Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarianHydrais one of the most basal metazoans and is a key model system for studying the peptides involved in these processes. We developed a novel peptidomic approach to the isolation and identification of functional signalling peptides fromHydra(theHydrapeptide project). First, peptides extracted from the tissue ofHydra magnipapillataare purified to homogeneity using high-performance liquid chromatography (HPLC). The isolated peptides are then tested for their ability to alter gene expression inHydrausing differential display-PCR (DD-PCR). If gene expression is altered, the peptide is considered as a putative signalling peptide and is subjected to amino acid sequencing. Following the sequencing, synthetic peptides are produced and compared to their native counterparts by HPLC and/or mass spectrometry (MS). The synthetic peptides, which are available in larger quantities than their native analogues, are then tested in a variety of biological assays inHydrato determine their functions. Here we present our strategies and a systematic approach to the identification and characterization of novel signalling peptides inHydra. We also describe our high-throughput reverse-phase nano-flow liquid chromatography matrix-assisted laser desorption ionization time-of-flight mass spectrometry (LC-MALDI-TOF-MS/MS) approach, which was proved to be a powerful tool in the discovery of novel signalling peptides.
