Processing of neuropeptide Y and somatostatin in human cerebrospinal fluid as monitored by radioimmunoassay and mass spectrometry.
The processing of four neuropeptides, neuropeptide Y (NPY) 1-36, NPY (18-36), somatostatin (SOM) 1-28, and SOM (15-28) was studied in human cerebrospinal fluid (CSF) by using a novel combination of methods that included radioimmunoassay (RIA) and mass spectrometry. Untreated CSF samples were chromatographed using reversed-phase high pressure liquid chromatography (RP-HPLC) followed by NPY-RIA or SOM-RIA. These results were compared with those obtained by incubating CSF with exogenous synthetic peptides and directly detecting peptide fragments by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). Using this combination of methods, we were able to determine the probable identities of peptides/peptide fragments recognized in radioimmunoassays. The most important NPY-immunoreactive components in CSF were found to be NPY (1-36) and NPY (3-36). Metabolic products of SOM (15-28) were found to contribute to SOM-like immunoreactivity (SOM-LI) in CSF, but SOM (1-28) only to a lesser degree. Differences in the rate of neuropeptide processing were observed. These differences depended more on the length of the peptide than its sequence. NPY (18-36) and SOM (15-28) were rapidly and extensively processed, whereas NPV (1-36) and SOM (1-28) were processed much more slowly in CSF. The production of SOM (15-28) from SOM (1-28) by enzymes in CSF was not observed. Also, the presence of a disulfide bond in the somatostatins appeared to stabilize them against enzymatic digestion of the ring structure. The results detailed in this report confirm MALDI-MS important role in studies of neuropeptide processing in CSF.
Nilsson
C
C
Institute of Clinical Neuroscience, Department of Neurochemistry, Göteborg University, Sahlgrenska University Hospital/Mölndal, Sweden. carol.nilsson@ms.se
Westman
A
A
Blennow
K
K
Ekman
R
R
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008970:Molecular Weight; D009478:Neuropeptide Y / Q000134:cerebrospinal fluid / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay / Q000379:methods; D013004:Somatostatin / Q000134:cerebrospinal fluid / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013997:Time Factors
D000596:Amino Acids; D009478:Neuropeptide Y; D010446:Peptide Fragments; D013004:Somatostatin; D000626:Aminopeptidases