Extensive Characterization of Tupaia belangeri Neuropeptidome Using an Integrated Mass Spectrometric Approach

Abstract

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

Broad characterization of endogenous peptides in the tree shrew visual system

“…Our methodological approach utilizes on the integration of data dependent acquisition (DDA) and target-mass list modes, with a mass list based on the previous DDA runs and also on the analysis of peptide rich areas of T. belangeri, i.e. striatum, performed previously [36]. Using this integrated approach, we achieved an identification of 52 peptides from 21 pro-hormone families, including 11 proenkephaline (PENK), 6 Somatostatin (SOM) and three Pro-MCH peptides.…”

“…Secondly, we used a targeted LC-MS/ MS analysis, with an inclusion list integrated from two independent sources: i) the list of peptides identified in the first step using the DDA approach ii) the previous findings by Petruziziello et al [36] performed on tree shrew striatum (peptide sequence FDR < 1%). The final workflow is shown in the Fig.…”

High Identification Rates of Endogenous Neuropeptides from Mouse Brain

“…Further comparative analysis shows that our new approach allowed more peptides to be identified than DDA LC-FT-MS/MS analysis combined with the commonly acetic acid solution (AAS) sample treatment method 2,23 or mixing in solution (MIS) multistage extraction method. 10 The results showed that about 82.0% of the peptides identified using method AAS +DDA had sequences identical with those using MOC+CSD. The peptides identified using method MIS+DDA and MOC+DDA had 83.1 and 84.7% sequences identical with those using MOC+CSD, respectively ( Figure 2B).…”

Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

“…The main advantage of this approach is that it allows the simultaneous monitoring of many peptides from the same brain tissue derived from a single drug protocol. We used a combination of a robust sample preparation method (18), high accuracy LC-MS analysis (19,20), and the use of multiple synthetic internal standards (21) to compare peptide levels in the DS between chronic nicotine and control animals. Our peptidome analysis determined 14 peptides exhibiting significant changes following chronic nicotine administration.…”