Absolute Quantification of Proteins in the Eye ofDrosophila melanogaster
Absolute Quantification of Proteins in the Eye ofDrosophila melanogaster
复制标题
DOI：
10.1002/pmic.201900049
复制
发表时间：
2020-09-03
期刊：
影响因子：
3.4
通讯作者：
Shevchenko, Andrej
中科院分区：
文献类型：
--
作者：
Raghuraman, Bharath Kumar;Hebbar, Sarita;Shevchenko, Andrej
蛋白质的绝对(摩尔)定量决定了它们在络合物、网络和代谢途径中的摩尔比例。利用MS Western工作流程，利用一个264 kDa的嵌合蛋白质标准，确定了果蝇眼睛中可能对形态发生和光转导(PT)至关重要的蛋白质的摩尔丰度，该标准总共涵盖了43种蛋白质中的197个肽。大多数蛋白质被两到四个蛋白型多肽独立定量，变异系数小于15%，优于1000倍的动态范围和亚毫微摩尔灵敏度。在这里，测定了野生型苍蝇眼睛和表现出扰动横纹肌形态发生的Incrumbs(CRB)突变眼的PT机械和横纹肌层(光敏细胞器)的蛋白质的摩尔丰度。
Absolute (molar) quantification of proteins determines their molar ratios in complexes, networks, and metabolic pathways. MS Western workflow is employed to determine molar abundances of proteins potentially critical for morphogenesis and phototransduction (PT) in eyes ofDrosophila melanogasterusing a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. The majority of proteins are independently quantified with two to four proteotypic peptides with the coefficient of variation of less than 15%, better than 1000-fold dynamic range and sub-femtomole sensitivity. Here, the molar abundance of proteins of the PT machinery and of the rhabdomere, the photosensitive organelle, is determined in eyes of wild-type flies as well as incrumbs(crb) mutant eyes, which exhibit perturbed rhabdomere morphogenesis.