蛋白质组学技术筛选与鉴定鲤鱼嗅脑急性创伤后的应激蛋白质

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

MALDI-TOF质谱技术研究海兔卵产卵激素及其分解产物的特性

采用萃取法和反相HPLC技术分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)卵内的产卵激素(egg laying hormone,ELH)及其分解产物.选用基质辅助激光解吸离子化飞行时间(Matrix-assisted laser deposition/ioni-zation time-of-flight,MALDI)质谱技术研究NLCS卵内的ELH、ELH分解产物和酸性多肽(acidic peptide,AP)二聚体的组成与结构特性.实验结果表明,NLCS卵不仅含有丰富的ELH及其分解产物,而且它的AP及其分解产物主要呈二聚体状态,推测这种二聚体结构对今后阐明ELH和AP的生理功能和调控机理起着重要的作用

质谱技术研究海兔大脑神经节超微量多肽内切酶和酸性多肽酶解产物

采用ESI-Q-TOF质谱分析了由27个氨基酸残基组成的酸性多肽(AP)的一级结构。选用RP-HPLC和MALDI-TOF质谱非在线技术分离与鉴定海兔大脑神经节(CG)多肽与蛋白质的组成与分布,发现CG中含有AP酶解的二聚体短肽。这些短肽序列分别为2(NKDEEQRELLKAISNLL)、2(NKDEEQRELLKAISNL)、2(SGVSLLTSNKDEEQREL)和2(LTSNKDEEQRE LL),均以L—R(氨基酸残基)方式断裂。以AP为探针,结合MALDI-TOF质谱分析技术,发现CG中含有超微量的L—R多肽内切酶,其分子量为78218·25Da。本研究中的分析方法也适合于研究其他生物体内超微量多肽及多肽酶分布与功能

优化分离与鉴定蓝斑背肛海兔口腔神经节蛋白质组

采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide m ass fin-gerprint,PMF)图谱.经数据库检索与比对后,发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率,它们分别是微管蛋白(Tubu lin)、肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(R ibu lose-1,5-b i-sphosphate carboxylase/oxygenase,R ibu lose),均属于神经细胞骨架蛋白质;同时还发现一种交配信号肽前体(Peptide m ating pheromone precursor).利用LOC trees软件和分类法对56种蛋白质进行亚细胞定位与分类

基质辅助激光解吸电离飞行时间质谱技术研究人血清转铁蛋白稳定性及裂解产物

制备质谱纯人血清转铁蛋白(HTF),供分子结构分析。选用SDS-PAGE、胶外酶解、基质辅助激光解吸/电离质谱技术(MALDI-TOF)、数据库检索和比对技术鉴定铁饱和HTF、双铁HTF(HTF-2Fe3+)、单铁HTF(HTF-Fe3+)和脱铁HTF(apoHTF)的稳定性和裂解产物。以乙腈溶液作为洗脱相,发现铁饱和HTF在RP-HPLC分离纯化过程中产生裂解现象。铁饱和HTF和HTF-2Fe3+经乙腈处理后均能产生不同分子量的短肽裂解产物,指出HTF结构稳定性与络合铁离子数量有关。铁组分改善了HTF分子结构的稳定性。采用比对法,研究在乙腈作用下HTF裂解成为各种各样短肽的规律,初步阐明其裂解机理。在乙腈作用下,HTF可能通过蛋白质去折叠途径,形成不同多聚态HTF或多肽裂解产物。推测目前用于临床诊断先天性糖基化紊乱(CDG)和慢性酒精滥用(CAA)疾病低准确率的起因可能是受HTF裂解产物或多聚体的干扰

Study of exceed micro-endopeptidease and enzymolysis produces of acidic peptide from the Aplysia cerebral ganglion by mass spectrometry

Acidic peptide (AP) consisting of 27 residues of amino acids was synthesized by the peptide synthetic instrument, and its primary structure was further analyzed with electrospray ionization-Q-time of flight (ESI-Q-TOF) mass spectrometer. The composition and distribution of the peptides and proteome in Aplysia cerebral ganglion were separated and identified by a combined off-line technology of reversed phase-high performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization (MALDI)-TOF mass spectrometry, respectively. A lot of enzymolysis produces of AP in cerebral ganglion(CG) were found, which showed a dipolymer molecular structure. It was noticed that the primary structure of these peptides were found to share a similar dipolymer structures with 2(NKDEEQRELLKAISNLL) 2 (NKDEEQRELLKAISNL), 2(SGVSLLTSNKDEEQREL), and 2(LTSNKDEEQRELL) and to have same L-R(amino acids) residues of bound split. Using a combined method of AP as an probe and MALDI-TOF mass spectrometry as a analytical technology, it was found that there was the exceed micro-endopeptidease for enzymolysis L-R bound of AP in CG, indicating that its molecular weight was 78218.25 Da. In addition, the analytical method described here is also fit for finding composition and distribution of the exceed micro-peptide and endo-peptidease in various organisms

Functions of Double Subunits of a Type,Structure of Iron Corn, and Kinetics of Iron Release from Membrane Ferritin of Human Placenta

E-mail: hqhuang@ xmu.edu.cn[中文文摘]以人胎盘组织为实验材料,小批量制备电泳纯人胎盘膜铁蛋白(HPMF),对其结构与功能进行研究。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术揭示,HPMF蛋白壳由分子量分别为15 kDa(MF15)和20 kDa(MF20)的亚基组成,其中MF15蛋白含量约为MF20的3倍。经肽质量指纹图谱(PMF)技术鉴定,发现PHMF的MF15和MF20亚基与人铁蛋白(HF)L亚基均具有较高的同源性,提出HPMF由单类型但分子量不同的双亚基组成的新观点。在选用基质辅助电离激光解吸飞行时间质谱(MALDI-TOF MS)技术直接分析HPMF亚基稳定性过程中,获得5个质荷比(m/z)值为5071.25,9962.51,15131.55(MF15),19936.40(MF20)和20147.61的特征质谱峰,其对应的亚基分子式分别为[MF15]3+,[MF20]2+,[MF15]+,[MF20]+和[MF20+Fe]+。ICP-MS分析发现,HPMF铁核中的铁和无机磷酸盐(Pi)含量仅为85Fe3+/HPMF和15Pi/HPMF,其中Fe3+/Pi比值约为5.72,明显低于绝大多数哺乳动物铁蛋白,但却高于细菌铁蛋白(BF)。在释放铁动力学方面,HPMF以一级反应动力学途径释放完整铁核中的铁量,其释放时间约需750 min,其释放铁的速率慢于马脾铁蛋白(HSF)和猪胰铁蛋白(PPF)(约60 min)。根据HPMF完全不同于大部分哺乳动物、植物和细菌铁蛋白的新颖结构与功能,提出HPMF在母体和胎儿之间中起着释放、储存和转运铁的复合生理功能的铁转运模型。[英文文摘]As an experimental material of human placental tissue，membrane ferritin of human placenta (HPMF) with electrophoresis purity was prepared in batch． SDS-PAGE approach reveals that the protein shell consists of double subunits of a type in HPMF，which the molecular weights of both subunits were calculated to be approximately 15 and 20 kDa respectively，named MF15 and MF20． In addition，the protein content of MF15 is about three times more than that of M20 in SDS-PAGE gel． Using the approach of peptide mass fingerprinting (PMF) ，both MF15and MF20 subunits were identified to show the high homology with reference to L subunit of human ferritin，pointing out that HPMF was a novel ferritin of two subunits involving in single type and different molecular weight． Using a matrix-assisted laser desorption ionization time of flight mass spectrometry to investigate the subunit-stability of HPMF，five mass peaks corresponding ratio (m/z) of mass to charge such as 5071.25,9962.51,15131.55 (MF15),19936.40 (MF20) and 20147.61 were indicated to have five subunit formula of [MF15]3 +,[MF20]2 +，[MF15]+，[MF20]+ and [MF20 + Fe]+ ，respectively． Using inductively coupled plasma-mass spectrometry to analyze the elemental composition of HPMF，only 85 Fe3 + and 15 inorganic phosphate (Pi) in the iron core of per molecular of HPMF were found，which was significantly lower than that of most mammal ferritins，but higher than that of bacterial ferritin ( BF) ． Kinetic studies showed that iron release from the complete HPMF followed the law with first-order reaction，which also differed from that with two different rates in mammal ferritins． In addition，the time of the iron release completely needed about 750 min in human placental membrane ferritin ( HPMF) ，which was longer than 60 min in both horse spleen ferritin ( HSF) and pig pancreatic ferritin ( PPF) , Accordingly,based on the difference of structure and function among HPMF，mammalian ferritins，plant ferritins，and BF，we proposed M15 and M20 subunit play different role in iron transporting from maternal blood to fetal blood．国家自然科学基金(No.30870515);973项目(No.2010CB12640)资

Optimized separation and identification of proteome from the baccal ganalion of Aplysia(Notarcus leachii cirrosus Stimpson)

A separation method of proteome from the buccal ganglion, of aplysia (Notarcus leachii cirrosus Stimpson, NLCS) with two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) was optimized, gaining about 300 protein spots. Mass spectrogram of peptide mass fingerprint(PMF) of 96 protein spots from the BG were obtained by a combined off-line technique of the matrix-assisted laser desorption ionization mass spectrometry(MALDI-TOF MS) and the emzymolysis in gel. Based on the identification via the databases searches and PMF maps, we found that four of 96 proteins in the gel showed the characteristics with high match scores, which were indicated to be framework proteins tubulin, actin, ribulose-1, 5-bisphosphate carboxylase and oxygenase in neurons cell; meanwhile, a peptide mating pheromone precursor was found. Fifty-six proteins in BG were further classified by a LOC tree software according to the search result of subcellular localization

Functions of Double Subunits of a Type, Structure of Iron Corn, and Kinetics of Iron Release from Membrane Ferritin of Human Placenta

As an experimental material of human placental tissue, membrane ferritin of human placenta (HPMF) with electrophoresis purity was prepared in batch. SDS-PAGE approach reveals that the protein shell consists of double subunits of a type in HPMF, which the molecular weights of both subunits were calculated to be approximately 15 and 20 kDa respectively, named MF(15) and MF(20). In addition, the protein content of MF(15) is about three times more than that of M(20) in SDS- PAGE gel. Using the approach of peptide mass fingerprinting (PMF), both MF(15) and MF(20) subunits were identified to show the high homology with reference to L subunit of human ferritin, pointing out that HPMF was a novel ferritin of two subunits involving in single type and different molecular weight. Using a matrix-assisted laser desorption ionization time of flight mass spectrometry to investigate the subunit-stability of HPMF, five mass peaks corresponding ratio (m/z) of mass to charge such as 5071. 25, 9962. 51, 15131. 55 (MF(15)), 19936. 40 (MF(20)) and 20147. 61 were indicated to have five subunit formula of [MF(15)](3+), [MF(20)](2+), [MF,](+), [MF(20)](+) and [MF(20+Fe)](+) respectively. Using inductively coupled plasma-mass spectrometry to analyze the elemental composition of HPMF, only 85 Fe(3+) and 15 inorganic phosphate (P(i)) in the iron core of per molecular of HPMF were found, which was significantly lower than that of most mammal ferritins, but higher than that of bacterial ferritin (BF). Kinetic studies showed that iron release from the complete HPMF followed the law with first-order reaction, which also differed from that with two different rates in mammal ferritins. In addition, the time of the iron release completely needed about 750 min in human placental membrane ferritin (HPMF), which was longer than 60 min in both horse spleen ferritin (HSF) and pig pancreatic ferritin (PPF). Accordingly, based on the difference of structure and function among HPMF, mammalian ferritins, plant ferritins, and BF, we proposed M(15) and M(20) subunit play different role in iron transporting from maternal blood to fetal blood