Technological advances in the proteomics has been quite remarkable. However, there remains difficulties in analyzing membrane proteins, peptides, minor proteins and functional state of proteins. In particular, peptides are hard to be detected with either two-dimensional electrophoresis or shotgun method. Furthermore, Such modification as oxidation and/or phosphorylation of peptides would surely affect their ionization efficiencies and quantitation of the modified peptides by mass spectroscopy would be quite difficult.The aim of our study is to search for peptides disease-specifically modified in the amount and functional states. Here, the peptides imply low molecular weight proteins including not only biologically active peptides but also cleaved ones. To achieve our purpose, we developed a method to successfully separate peptides in crude extracts of tissues with high yield and reproducibility. This method enables us to compare quantitatively amount of each peptide in normal and disease tissues. We applied the method to extraction of peptides in renal cortex of the diabetic (db/db) and its control mice (+/+). The peptides of three db/db mice were analyzed by MALDI TOF-MS, and their spectra were compared with those of three +/+ mice. The results showed that two peptides were changed in amounts due to diabetes mellitus. We have succeeded in purifying these peptides by two-dimensional HPLC and identifying them by amino acid sequencing.
