Peptidome database is aimed to accumulate the fact data for endogenous peptides based on their physicochemical properties including hydrophobicity, net charge and molecular weight. To construct this database, the crude peptide fraction was first subjected to gel filtration. Two peptide fractions of Mr<3,000 and 3,000<Mr<6,000 were each separated into 5,000 fractions by 2-dimensional (2D) HPLC (1st strong cation exchange (SCX) and 2nd reverse phase (RP) HPLC), and an aliquot of each fraction was analyzed with MALDI-TOF, ESI-Q-TOF and MALDI-TOF-TOF mass spectrometers (MSs). Starting from 1-2 g of tissue, we detected 16,788 peptides of Mr<6,000 in the pig brain and 4,058 peptides of Mr<3,000 in the mouse brain, and 26% of the peptides were identified with the tandem MSs. Among the identified peptides, about 20% were derived from peptide precursor proteins or secretory proteins, but about 30% were considered to be generated by degradation of the proteins. The biological activity of each fraction on the 2D-HPLC is also being evaluated and stored in the database. By combining these data, Peptidome database can be used as the powerful and intellectual basement for the discovery of new peptides.
We are also developing the efficient and quick peptide information acquisition system, which consists of automated 2D-HPLC (step-wise/linear and linear/linear) coupled on-line with ESI-TOF MS and MALDI-TOF-TOF MS. Three-fourths of the eluates from the 2D-HPLC were infused into the ESI-TOF MS, and the remaining portion was spotted on the target plates for MALDI-TOF/TOF MS. In the preliminary analysis, about 800 peptides and 160 peptides were detected and identified as for the pig brain peptides of Mr<3,000 obtained from 1.5 g of tissue.
