Improved identification of endogenous peptides from murine nervous tissue by multiplexed peptide extraction methods and multiplexed mass spectrometric analysis.

In recent years, mass spectrometry (MS) based techniques have made their entrance in the analysis of endogenous peptides extracted from nervous tissue. In this study, we introduce a novel peptide extraction procedure using 8 M urea, next to the more established extraction method that uses acetic acid. The extracted peptide mixtures are analyzed by both high-resolution nanoLC MS/MS using collision induced dissociation (CID) on an LTQ-Orbitrap and nanoLC electron transfer induced dissociation (ETD) on a linear ion trap. The combined use of the two extraction methods significantly increased the yield of identified endogenous neuropeptides. The multiplexed use of high mass accuracy mass spectrometry and the ETD fragmentation technique further increased the yield and confidence of peptide identifications. Furthermore, reduction of disulfide bridges during sample preparation was essential in the identification of several endogenous peptides containing cysteine disulfide bonds. Through this study, we identified in total 142 peptides in extracts of the mouse pituitary tissue, whereby 43 uniquely in the urea extract and 11 uniquely in the acetic acid extract. A large number of detected endogenous peptides were reported previously, but we confidently identified 22 unreported peptides that possess characteristics of endogenous peptides and are thus interesting targets to be explored further.