Amidation/non-amidation top-down analysis of endogenous neuropeptide Y in brain tissue by nano flow liquid chromatography orbitrap Fourier transform mass spectrometry.

Neuropeptide Y (NPY) is a transmitter molecule in nerve system, and it was an over 4-kDa large peptide with the C-terminal end amidation. NPY is biosynthesized through many maturation processes from a large pre-pro-peptide with peptide-cleavages and amidation that is important to study the biosynthesis regulation. Previously, it was reported that cathepsin L participates in the production of NPY and that cathepsin L generates both of amidated and non-amidated NPYs. However, the non-amidated NPY (NPY-COOH) has not been reported in brain tissues until now. In this study, endogenous NPY-COOH in mouse brain tissue was detected and identified by using nano flow liquid chromatography (nanoLC) orbitrap Fourier transform mass spectrometry (FT-MS) after the effective purification and separation of NPY-COOH from NPY-amide and other peptides using two different gel-filtration chromatography. Amidated NPY was eluted earlier than non-amidated NPY-COOH in the C18 reversed phase nanoLC and the silica-based gel-filtration chromatogram with hydrophobic interaction. The amount of endogenous NPY-COOH was about 0.05% of the matured NPY-amide amount in adult mouse brain.