优化建立鲤鱼(cyprinus carpio,CC)嗅觉端脑(嗅脑)全蛋白提取技术。联用低渗透裂解和液氮冻溶法破碎鲤鱼嗅脑组织(rhinencephalon tissue of cyprinus carpio,CCRT)、低速离心提取CCRT全蛋白,并采用双向凝胶电泳(2D-PAGE)技术进行有效分离。经分析与统计,每张CCRT的2D-PAGE图谱中的蛋白质斑点数目约为1200个。分别分离CCRT的脂溶性和水溶性全蛋白,并获得高分辨率的2D-PAGE图谱。选用差异蛋白质组学技术筛选经10%冰醋酸创伤后的CC,其端脑组织所表达出的6种应激蛋白质,并用肽质量指纹谱(peptide mass fingerprinting,PMF)和数据库检索技术给予鉴定。其中3种蛋白质为70S热休克蛋白、β微管蛋白和DNA链接酶IV,有望作为研究大脑急性创伤后的应激修复途径和机理的指示蛋白质。
Pig pancreas ferritin(PPF) was purified by ultra-centrifugation, ion-exchange chromatography, and native gradient polyacrylamide gel electrophoresis(PAGENG). Sodium dodecyl sulfate(SDS)-PAGE indicates that PPF consists of two subunit types, namely, H(21000) and L(19000) subunits, and its core shows an average element composition of 1698 Fe3+ and 179 phosphate molecules within the hollow shell, giving a 9.5:1 ratio of Fe3+ to phosphate. An off line approach combining reversed-phase high-performance liquid chromatography(RP-HPLC) with matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS) made the decomposition of PPF shell into H and L subunits for the analysis of mass spectrometry(MS), giving molecular weights of both H(21014.4) and L(18319.9) subunits. Both subunit types were further identified by an approach combining peptide mass fingerprint(PMF) with database search. A ratio of 1H to 2L subunits in PPF was determined by SDS-PAGE, RP-HPLC, and MALDI-TOF MS, respectively. It is well known that the non-covalent interaction of L-L or H-L subunits is stronger than that of H-H subunits in PPF, which may be further used to explain the unclear physiological function between H and L subunits in PPF.
选用柱层析、电泳和反相高效液相色谱（RP-HPLC）技术制备质谱纯棕色固氮菌细菌铁蛋白（Bacteri-al ferritin ofAzotobacter vinelandii,AVBF）,并采用释放铁动力学和肽质量指纹图谱（Peptide mass fingerprint-ing,PMF）技术分别鉴定AVBF。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和电泳技术揭示AVBF亚基之间相互作用强度、稳定性和聚合态。AVBF可直接捕获有机小分子亚甲蓝（MB）,其捕获率为15.0±2.0MB/AVBF,认为介于AVBF亚基单体之间的血红素参与捕获MB。较高浓度（40%～50%）的乙腈和丙酮均能使AVBF和鲨鱼肝铁蛋白（Liver ferritin of shark,SLF）释放不稳定亚基,但在较低浓度（20%～30%）的乙腈条件下,却需要借助来源于质谱仪的激光才能使AVBF或SLF释放不稳定亚基,并供质谱分析。AVBF亚基之间的相互作用强度明显低于SLF。铁蛋白亚基之间的相互作用强度高低与铁蛋白执行释放和储存铁的速率有关。
