A simple method for peptide purification as a basis for peptidome analysis

Abstract

We developed a simple method for purifying peptides from crude extracts of animal tissues. Peptides free of high-molecular-mass (HMM) proteins were prepared by differential solubilization of proteins and peptides followed by tricine-SDS-PAGE. Proteins and pep-tides in a tissue extract were co-precipitated by slowly dropping the extracts into acetone at −40°C. The precipitates were dissolved in 2 mM DTT and separated by centrifugation. The peptide-enriched supernatant was resolved by tricine-SDS-PAGE and portions of the lanes below 10 kDa were excised. Peptides were extracted from the gel pieces in 40% ace-tonitrile and fractionated by two-step reversed-phase HPLC. We evaluated the method by extracting peptides from the livers and kidneys of rats and diabetic model mice. The HPLC elution profiles of peptide mixtures were reproducible, peptide recovery from tricine-SDS-PAGE gels was high and we identified one HPLC-fractionated peptide by amino-acid sequencing. The present method should serve as a good foundation for future peptidomics.