Lomazi Rafael L, Nishiduka Erika S, Silva Pedro I, Tashima Alexandre K
Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, SP, Brazil.
Methods Mol Biol. 2018;1719:359-367. doi: 10.1007/978-1-4939-7537-2_24.
Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.
蜘蛛毒液由数百种蛋白质和肽组成。其中几种毒液毒素是质量范围在3至9千道尔顿的富含半胱氨酸的肽。小肽（<3千道尔顿）可以通过质谱分析进行全面表征，而蛋白质通常通过自下而上的方法进行鉴定，即首先用胰蛋白酶消化蛋白质以产生较短的肽用于串联质谱表征。一般来说，对两种或更多种肽进行测序就足以鉴定蛋白质，但对于毒液肽组学而言，完全阐明肽序列以及分子中二硫键的数量是很有必要的。在本章中，我们描述了一种通过多重酶消化、天然肽和消化后肽的质谱分析、从头分析以及序列重叠比对来完全测序并确定质量范围在3至9千道尔顿的蜘蛛毒液肽中二硫键数量的方法。
