Biringer Roger G, Amato Heidi, Harrington Michael G, Fonteh Alfred N, Riggins James N, Hühmer Andreas F R
Scientific Instruments Division of Thermo Electron Corporation, 355 River Oaks Parkway, San Jose, CA 95134, USA.
Brief Funct Genomic Proteomic. 2006 Jun;5(2):144-53. doi: 10.1093/bfgp/ell026. Epub 2006 May 26.
The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan LTQ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications.
脑脊液（CSF）为了解中枢神经系统的健康状况提供了一个便捷途径，并且在脑部疾病中已记录到一些脑脊液蛋白质的变化。然而，蛋白质的完整种类尚不清楚，鉴定蛋白质成分的方法仍在不断发展。本研究的目的是检查用不同蛋白酶产生的人脑脊液消化物的序列覆盖率。使用精氨酸-C内肽酶（ArgC）、谷氨酸内肽酶（GluC）、胰凝乳蛋白酶、胰蛋白酶及其组合获得脑脊液蛋白质的酶解消化物，然后使用反相色谱法和Finnigan LTQ线性离子阱质谱仪进行检测。用BioWorks 3.1鉴定肽序列，并计算38种最可靠鉴定的蛋白质的序列覆盖率。胰蛋白酶和GluC产生的覆盖率高于胰凝乳蛋白酶，而ArgC的序列覆盖率最低。在四个数量级的丰度动态范围内，蛋白质序列覆盖率仅受到轻微影响。将来自不同蛋白酶的肽组合起来可进一步提高覆盖率。通过结合GluC和胰蛋白酶的消化结果实现了最大序列覆盖率。这些结果对未来鉴定脑脊液蛋白质及其翻译后修饰的研究具有启示意义。
