Shettima Abubakar, Ishak Intan H, Abdul Rais Syahirah Hanisah, Abu Hasan Hadura, Othman Nurulhasanah
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Gelugor, Pulau Pinang, Malaysia.
Department of Microbiology, Faculty of Science, University of Maiduguri, Maiduguri, Borno State, Nigeria.
PeerJ. 2021 Mar 3;9:e10863. doi: 10.7717/peerj.10863. eCollection 2021.
Proteomic analyses have broadened the horizons of vector control measures by identifying proteins associated with different biological and physiological processes and give further insight into the mosquitoes' biology, mechanism of insecticide resistance and pathogens-mosquitoes interaction. Female ingests human blood to acquire the requisite nutrients to make eggs. During blood ingestion, female mosquitoes transmit different pathogens. Therefore, this study aimed to determine the best protein extraction method for mass spectrometry analysis which will allow a better proteome profiling for female mosquitoes.
In this present study, two protein extractions methods were performed to analyze female proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBuster. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis.
The CytoBuster reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBuster reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBuster reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBuster protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.
蛋白质组学分析通过鉴定与不同生物和生理过程相关的蛋白质，拓宽了病媒控制措施的视野，并进一步深入了解蚊子的生物学特性、抗杀虫剂机制以及病原体与蚊子的相互作用。雌蚊吸食人类血液以获取制造卵子所需的营养物质。在吸血过程中，雌蚊会传播不同的病原体。因此，本研究旨在确定用于质谱分析的最佳蛋白质提取方法，以便更好地对雌蚊进行蛋白质组分析。
在本研究中，通过三氯乙酸丙酮沉淀提取法和商用蛋白质提取试剂细胞裂解液（CytoBuster），采用两种蛋白质提取方法分析雌蚊蛋白质组。然后，通过液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）进行蛋白质鉴定，并随后进行功能蛋白质注释分析。
与三氯乙酸丙酮沉淀提取法平均蛋白质产量为283.15微克相比，细胞裂解液试剂的蛋白质产量最高，平均为475.90微克。LC-ESI-MS/MS分别从细胞裂解液试剂和三氯乙酸丙酮沉淀法中鉴定出1290种和890种蛋白质。在比较两种方法中的蛋白质类别时，使用细胞裂解液试剂鉴定出的蛋白质还有另外三个类别。这些蛋白质与支架/衔接蛋白（PC00226）、蛋白质结合活性调节剂（PC0009）和细胞间信号分子（PC00207）有关。总之，就蛋白质产量、蛋白质组覆盖率和提取速度而言，细胞裂解液蛋白质提取试剂在蛋白质提取方面表现更佳。
