High Identification Rates of Endogenous Neuropeptides from Mouse Brain

Abstract

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC−MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.

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

“…We used the mixing on column method (19) . Before filtration, the first two-step aqueous extractions, and the last two-step organic extractions were mixed, respectively.…”

“…For each LC-MS analysis, the organic extract was loaded first on the trap column, and after a 3-min conditioning, the aqueous extract was loaded with the same volume (5 l). The usage of organic solvent in samples was demonstrated to be necessary to increase the recovery of large peptides in injection, separation, and storage (19). For all of the brain samples, the concentration of each internal standard was 20 nM in the final solution.…”

“…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.…”

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry

“…The mixing on column (MOC) method was used for sample treatment. 20 Briefly, each brain tissue was extracted 2 times using aqueous solution and 2 times using acetonitrile solution, respectively. In each step, 3 min of homogenization will be performed to assist the extraction.…”

“…We first extracted all peptides from a brain sample and then loaded them using a MOC method to increase the peptide extraction coverage. 29 One group of peptide extracts were then submitted to reduction and alkylation using DTT and IAM, respectively, as is commonly done in bottom-up proteomics applications. 30 As verified using peptide Melanin-concentrating hormone (MCH, DFDMLRCMLGRVYRPCWQV), which contains an intrapeptide disulfide bond, the alkylation rendered disulfide bonded peptides with increasing molecular mass and hydrophobicity but without changing their charge states (Figure 1).…”