One-Step Sampling, Extraction, and Storage Protocol for Peptidomics Using Dihydroxybenzoic Acid

Abstract

The isolation and extraction of natively occurring signaling peptides (SPs) from tissue is a critical first step in characterizing these peptides. Recent studies have outlined several approaches designed to preserve and extract SPs from tissue. Here, we demonstrate a surprisingly simple method to extract SPs from tissue samples, ranging from cell clusters to brain punches to intact brain regions, using a matrix often employed in matrix-assisted laser desorption/ionization mass spectrometry-2,5-dihydroxybenzoic acid (DHB). DHB allows for the effective extraction of endogenous peptides from tissue as well as long-term preservation of tissue samples and extracts. Using the mouse pituitary gland as a model, the extraction protocol effectively recovers 24 known and many additional putative peptides from individual samples. Peptide extracts stored in the DHB extraction media are stable for years without freezing. The approach is also effective for other neuronal tissues; the complement of neuropeptides in bag cell neuron clusters from the Aplysia central nervous system, the rat cerebellum, and rat dorsal striatum also have been examined. Advantages of this new extraction procedure are its technical simplicity, reproducibility, ease of remote preparation of samples, and long-term sample preservation without freezing.