周围神经再生条件液中蛋白条带质谱分析

ANALYSIS OF PROTEIN BAND IN PERIPHERAL NERVE REGENERATION CONDITIONED FLUID BY SHOTGUN TECHNIQUE

目的研究神经再生条件液（nerve regeneration conditioned fluid,NRCF）中具有神经再生趋化活性的蛋白成分,对相对分子质量为（232～440）×10^3的蛋白条带进一步行分离和定性。方法新西兰大白兔6只,体重1.8～2.5kg,取坐骨神经建立神经再生室模型。术后7d采集NRCF进行自然聚丙烯酰胺凝胶电泳（native-polyacrylamide gel electrophoresis,Native-PAGE）分离,切取相对分子质量为（232～440）×10^3区域的蛋白条带,采用Shotgun蛋白质组学策略、液相色谱-串联质谱对其组分进行定性分析。结果NRCF的Native-PAGE电泳结果显示,相对分子质量在669×10^3以上、（232～440）×10^3和（140～232）×10^3分别有1条蛋白带,在（67～140）×10^3有6条蛋白带。其中相对分子质量在（232～440）×10^3的蛋白条带共鉴定到54种蛋白质,主要集中在等电点5.5～8.0,相对分子质量为（10～40）×10^3范围内,其中4种是未命名蛋白质。这54种蛋白质可归类为结合蛋白（43%）、转运蛋白（30%）、酶（6%）、信号转导蛋白（4%）和分子功能未知（17%）。54种蛋白质的亚细胞定位主要是分泌性蛋白（63%）,其他的位于细胞膜和胞浆内等。结论采用Native-PAGE和Shotgun蛋白质组学分析NRCF中相关蛋白质,确定了NRCF中相对分子质量为（232～440）×10^3条带的蛋白质组分,为进一步研究其中的功能未知蛋白提供了线索,对明确其是否对周围神经再生具有促进作用。

Objective To study the biological activities of the nerve regeneration conditioned fluid （NRCF）, especially to further separate and identify the protein bands of the relative molecular mass of （232-440）×10^3. Methods The silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve in 6 New Zealand white rabbits （weight, 1.8-2.5 kg）. The proteins in NRCF were separated by the native-polyacrylamide gel electrophoresis （Native-PAGE）, the protein bands of the relative molecular mass of （232-440）×10^3 were analyzed by the Shotgun technique, liquid chromatography, and mass spectrometry. Results The Native-PAGE result showed that there was 1 protein band of the relative molecular mass over 669 × 10^3, （232-440） × 10^3 and （140-232）×10^3, respectively, and 6 bands of the relative molecular mass of （67-140）×10^3. Besides, 54 proteins were identified with at least 2 distinct peptides in 1 protein band of the relative molecular mass of （232-440） × 10^3 , including 4 unnamed protein products, mainly at the isoelectric points of 5.5-8.0 and of the relative molecular mass of （10-40） × 10^3. Based on their functions in the protein database, all the identified proteins in this study were classified into the following 5 groups： conjugated protein （43 % ）, transport protein （30 % ）, enzyme （6 % ）, signal transducer （4 % ）, and molecular functionunknown protein （17%）. At the subcellular localization of the identified proteins, there was mainly a secreted protein （63%）, and the remaining proteins were localized in the membrane and cytoplasm. Conclusion Native-PAGE and the Shotgun technique can effectively separate and identify proteins from NRCF, and can identify the components of the protein band of the relative molecular mass of （232-440） × 10^3 and provide basic information on the unnamed protein products in NRCF.