Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry
Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry
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DOI：
10.1016/j.ibmb.2006.10.011
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发表时间：
2007-02-01
影响因子：
3.8
通讯作者：
Willis, Judith H.
中科院分区：
文献类型：
--
作者：
He, Ningjia;Botelho, Julianne M. C.;Willis, Judith H.
已鉴定的表皮蛋白质的鉴定是基于从清洁的表皮中提取的单个蛋白质的分离和测序。这些数据促进了从cDNA或基因组序列的概念翻译的序列的分类。问题是，这些假定的表皮蛋白质是否真的被整合到表皮中。本文从冈比亚按蚊蜕皮过程中被自身清理的表皮蛋白质入手，对冈比亚按蚊表皮蛋白质进行了分析。采用SDS凝胶电泳对幼虫和蛹的头囊和蛹囊蛋白质进行了分离。将大的凝胶切片还原、脲甲基化并用胰蛋白酶消化。还用胰蛋白酶处理SDS提取后剩余的沉淀物。所得肽在C18柱上分离，然后通过串联质谱法进行分析。二百九十五肽从假定的表皮蛋白进行了鉴定，这些对应于最少69和最多119种不同的蛋白质。每一个报告作为一个真正的按蚊表皮蛋白的第一次。除了两个已知的角质层蛋白家族的成员，其他家庭的成员可能是角质层的结构组成部分进行了鉴定。此外，还鉴定了可归因于蜕皮液、肌肉和硬化剂的其他肽。(c)2006年由Elsevier Ltd.出版
Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents. (c) 2006 Published by Elsevier Ltd.