A peptidomics strategy for discovering endogenous bioactive peptides.
Peptide hormones and neuropeptides constitute an important class of naturally occurring peptides that are generated from precursor proteins by limited proteolytic processing. An important but unaddressed issue in peptidomics is to pin down novel bioactive peptides in a bulk of peptide sequences provided by tandem mass spectrometry. Here, we describe an approach to simultaneously screen for bioactive peptides and their target tissues. The principle behind this approach is to identify intact secretory peptides that have the ability to raise intracellular calcium levels. In practice, we used nanoflow liquid chromatography-tandem mass spectrometry to analyze peptides released by exocytosis from cultured cells. Peptide sequence information was utilized to deduce intact peptide forms, among which those highly conserved between species are selected and tested on an ex vivo calcium assay using tissue pieces from transgenic mice that systemically express the calcium indicator apoaequorin. The calcium assay can be applied to various cell types, including those not amenable to in vitro culture. We used this approach to identify novel bioactive neuropeptides derived from the neurosecretory protein VGF, which evoke a calcium response in the pituitary and hypothalamus.
Sasaki
Kazuki
K
Department of Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan. ksasaki@ri.ncvc.go.jp
Takahashi
Noriyuki
N
Satoh
Mitsuo
M
Yamasaki
Motoo
M
Minamino
Naoto
N
D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
D001059:Apoproteins; D009479:Neuropeptides; D010455:Peptides; D011994:Recombinant Proteins; C522413:VGF peptide; C060014:apoaequorin; D000331:Aequorin