基质辅助激光解吸／电离质谱法研究海兔卵多肽内切酶组成、结构与功能.docVIP

基质辅助激光解吸／电离质谱法研究海兔卵多肽内切酶组成、结构与功能 【摘要】选择DEAE52纤维素和Sephadex G150为分离介质,采用柱层析法分离纯化海兔卵多肽酶（Endopeptidease of Aplysia egg，AEE）。选用十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDSPAGE）法测定AEE纯度和亚基分子量(约39 kDa)。基质辅助激光解吸／电离（MALDITOF）质谱技术测定AEE由单类型亚基(M)组成，其m/z比值分别为12738.17、18108.79和38221.42，即分子式为\[M3+\]、\[M2+\]和\[M+\]。其中AEE分子量为38221.42 Da (称为AEE39)，是一种含金属锌的多肽酶。以胰岛素(INS)为探针,研究AEE39酶切INS能力,其酶切产物m/z比值为1449.51、2085. 84、40 80.41和4165.42。自行设计INS序列分析软件，鉴定AEE39酶切INS的位点，发现易酶切位点是LeuX (氨基酸残基)，其次是GluX，部分PhenX、AsnX和SerX结构也可以发生酶切。通过INS和海兔吸引素的分子结构比对后，指出海兔卵中的AEE39主要功能之一是负责酶切海兔吸引素，起着海兔之间信息交往、召唤、识别和交配的重要作用。AEE39具有酶切酸性多肽（acidic peptide, AP）中LeuLeu的能力，属于一种广谱性多肽内切酶。 【关键词】 海兔，多肽内切酶，质谱分析，吸引素，胰岛素 Composition, Structure and Function from Endopeptidease of Aplysia Ｅgg　Ａnalyzed with Matrix Assisted Laser Desorption IonizationTime　of FlightMass Spectrometry Huang HeQing, Lu YongJin, Lin QingMei, Zhuo HuiQin, Huang HuiYing 1(Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, 2State Key Laboratory of Physical Chemistry of Solid Surface, Collage Chemistry Chemical Engineering, 3State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005) Abstract　Endopeptidease of Aplysia egg (AEE) was separated and purified by column chromatography using separation gel both DEAE52 cellulose. The purity of AEE and its molecular weight of the subunit (approximately 39.0 kDa) were determined by Sephadex G150 and SDSpolyacrylamid gel electrophoresis (PAGE) methods. The results from MALDITOF mass spectrometry indicated that AEE39 consisted of single subunit type (M), and its ratio of mass to charge (m/z) was 12738.17, 18108.79 and 38221.42, called [M3+], [M2+], and [M+], respectively. Using probe of insulin (INS), a combined technology with both metal chelator EDTA and MALDITOF mass spectrometry was employed to measure molecular weight of AEE３９ and to identify an endopeptidease with metal zinc, s