Nilsson C, Westman A, Blennow K, Ekman R
Institute of Clinical Neuroscience, Department of Neurochemistry, Göteborg University, Sahlgrenska University Hospital/Mölndal, Sweden.
Peptides. 1998;19(7):1137-46. doi: 10.1016/s0196-9781(98)00071-0.
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.
通过使用包括放射免疫分析（RIA）和质谱分析在内的新方法组合，对人脑脊液（CSF）中四种神经肽，即神经肽Y（NPY）1 - 36、NPY（18 - 36）、生长抑素（SOM）1 - 28和SOM（15 - 28）的加工过程进行了研究。未处理的脑脊液样本先采用反相高压液相色谱法（RP - HPLC）进行色谱分离，然后进行NPY - RIA或SOM - RIA。将这些结果与通过将脑脊液与外源性合成肽孵育并通过基质辅助激光解吸/电离飞行时间质谱（MALDI - MS）直接检测肽片段所获得的结果进行比较。使用这种方法组合，我们能够确定放射免疫分析中识别出的肽/肽片段的可能身份。脑脊液中最重要的NPY免疫反应性成分被发现是NPY（1 - 36）和NPY（3 - 36）。发现SOM（15 - 28）的代谢产物对脑脊液中的SOM样免疫反应性（SOM - LI）有贡献，但SOM（1 - 28）的贡献较小。观察到神经肽加工速率存在差异。这些差异更多地取决于肽的长度而非其序列。NPY（18 - 36）和SOM（15 - 28）被快速且广泛地加工，而NPV（1 - 36）和SOM（1 - 28）在脑脊液中的加工速度要慢得多。未观察到脑脊液中的酶将SOM（1 - 28）加工成SOM（15 - 28）。此外，生长抑素中存在的二硫键似乎使其对环结构的酶促消化具有稳定性。本报告中详细阐述的结果证实了MALDI - MS在脑脊液神经肽加工研究中的重要作用。
