Comparison of extraction methods for peptidomics analysis of mouse brain tissue

The peptidome encompasses all the peptides present in a particular cell, tissue or organism at a particular point in time. Neuropeptidomics studies the peptidome of the nervous system and will become increasingly important in neuroscience research. Novel peptides can be discovered and, when applied to disease models, key players in pathophysiological mechanisms will be identified. That way, they can serve as drug targets or biomarkers. Presently, different extraction protocols are in use, but no consensus has been reached on what fixation and extraction protocol is best suited for brain tissue. Therefore, in this article we compare different methods for quenching of proteolytic activity (snap-freezing of whole mouse in liquid nitrogen immediately after cervical dislocation, freezing of the dissected brain in 2-methyl-butane and heat denaturation of the tissue by microwave treatment) in combination with different extraction methods. The protocol that combines submersion in liquid nitrogen with extraction in 0.25% acetic acid results in the highest number of unique identifications, a high conservation of posttranslational modifications, the best reproducibility between duplicate samples and the best comparison with former studies on mouse brain peptides. For these reasons, we recommend the use of this protocol in future neuropeptidomics studies.