Hansson Karl T, Skillbäck Tobias, Pernevik Elin, Holmén-Larsson Jessica, Brinkmalm Gunnar, Blennow Kaj, Zetterberg Henrik, Gobom Johan
Inst. of Neuroscience and Physiology, Dept. of Psychiatry and Neurochemistry, Sahlgrenska Academy at University of Gothenburg;
Inst. of Neuroscience and Physiology, Dept. of Psychiatry and Neurochemistry, Sahlgrenska Academy at University of Gothenburg; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital.
J Vis Exp. 2017 Dec 4(130):56244. doi: 10.3791/56244.
This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step. Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers. Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution. Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.
本方案描述了一种用于鉴定人脑脊液（CSF）中内源性肽的方法。为此，将先前基于分子量截留（MWCO）过滤和质谱分析开发的方法与离线高pH反相HPLC预分级步骤相结合。分泌到脑脊液中是中枢神经系统细胞释放的分子清除的主要途径。因此，中枢神经系统中的许多过程都反映在脑脊液中，使其成为一种有价值的诊断液。脑脊液成分复杂，含有浓度范围跨越8-9个数量级的蛋白质。除蛋白质外，先前的研究还证明了大量内源性肽的存在。虽然对内源性肽的研究不如对蛋白质广泛，但它们作为生物标志物也可能具有潜在的研究价值。通过MWCO过滤将内源性肽与脑脊液中的蛋白质成分分离。通过去除样品中的大部分蛋白质成分，可以增加所研究的样品体积，从而也增加内源性肽的总量。在LC-MS分析之前，通过在碱性pH下进行反相（RP）HPLC预分级步骤来解决过滤后肽混合物的复杂性问题。分级与一种简单的合并方案相结合，将60个级分合并为12个级分，从而可以减少分析时间消耗，同时仍能在很大程度上避免共洗脱。使用三种不同的肽/蛋白质鉴定软件程序进行自动肽鉴定，随后合并结果。不同的程序是互补的，而不是可比较的，三种程序之间的鉴定重叠不到15%。
