In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis
In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis
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DOI：
10.1021/ac9907181
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发表时间：
1999-12-15
影响因子：
7.4
通讯作者：
Sweedler, JV
中科院分区：
文献类型：
--
作者：
Li, LJ;Garden, RW;Sweedler, JV
探索了使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）与源后衰变（PSD）和碰撞诱导解离（CID）碎片离子质量分析直接测序来自生物细胞的肽的能力。描述了三种不同的样品制备方法，用于对来自无脊椎动物模型Aesthesia californica的组织样品和单个神经元中的肽进行测序。为了表征来自心房腺的肽，将MALDI-PSD/CID直接应用于覆盖有基质α-氰基-4-羟基肉桂酸（CHCA）的组织印迹。所得的碎片离子结合数据库搜索确认了几个新的产卵激素基因编码的肽的结构。此外，一个单一的身份不明的神经元的MS分析检测与肌调节蛋白C和E的分子量的肽;这一分配确认使用MALDI-PSD与基质2，5-二羟基苯甲酸（DHB）。DHB并不总是为PSD实验提供足够的片段化;因此，开发了采用DHB和CHCA的独特的双基质取样方法，以直接测序来自单个脑神经节B细胞的十肽。来自细胞样品的碎片离子的质量准确度对于所采用的仪器是典型的，并且不受样品的形态和复杂性的不利影响。
The ability to directly sequence peptides from biological cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with postsource decay (PSD) and collision-induced dissociation (CID) fragment ion mass analysis is explored. Three different sample preparation methods are described for sequencing peptides in tissue samples and in single neurons from the invertebrate model Aplysia californica. To characterize peptides from the atrial gland, MALDI-PSD/CID is applied directly to a tissue blot covered with the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The resulting fragment ions combined with database searching confirm the structure of several novel peptides encoded by egg-laying hormone genes. Moreover, MS profiling of a single unidentified neuron detects peptides with molecular weights of myomodulins C and E; this assignment is confirmed using MALDI-PSD with the matrix 2,5-dihydroxybenzoic acid (DHB). DHB does not always provide adequate fragmentation for PSD experiments; therefore, a unique dual-matrix sampling method, employing both DHB and CHCA, is developed to directly sequence a decapeptide from a single cerebral ganglion B cell. Mass accuracy of fragment ions from cellular samples is typical for the instrument employed and is not deleteriously affected by the morphology and complexity of the samples.