Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC.
Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.
Joseph
Tessy
T
Department of Anaesthesia, Faculty of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074, Singapore.
Lee
Tat Leang
TL
Ning
Chou
C
Nishiuchi
Yuji
Y
Kimura
Terutoshi
T
Jikuya
Hiroyuki
H
Ou
Keli
K
Chin
Yau Chin
YC
Tachibana
Shinro
S
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000134:cerebrospinal fluid / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018847:Opioid Peptides / Q000037:antagonists & inhibitors / Q000134:cerebrospinal fluid* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D010146:Pain / Q000378:metabolism / Q000503:physiopathology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000134:cerebrospinal fluid / Q000302:isolation & purification / Q000502:physiology; D011498:Protein Precursors / Q000134:cerebrospinal fluid* / Q000302:isolation & purification* / Q000378:metabolism; D011863:Radioimmunoassay; D011957:Receptors, Opioid / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097629:Nociceptin
D008715:Methionine; D009479:Neuropeptides; D018847:Opioid Peptides; D020033:Protein Isoforms; D011498:Protein Precursors; D011957:Receptors, Opioid; D000097629:Nociceptin; C111148:nocistatin; C119224:pronociceptin