Aix-Marseille Univ., IRD, SSA, AP-HM, VITROME, Marseille, France.
IHU Méditerranée Infection, Marseille, France.
Parasit Vectors. 2022 Jun 30;15(1):240. doi: 10.1186/s13071-022-05361-0.
In the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling.
Three homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes.
Relevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database.
The results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.
在过去的十年中，基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的创新方法已经出现，用于节肢动物鉴定。越来越多的人有兴趣将原始技术应用于节肢动物鉴定，这导致了各种用于样品制备和选择身体部位的程序的发展。然而，缺乏共识策略阻碍了直接的研究间比较。此外，这些不同的程序对新用户来说令人困惑。因此，有必要为 MALDI-TOF MS 鉴定建立优化程序和标准化协议，这将显著促进参考 MS 数据库的共享。在这里，我们评估了使用 MALDI-TOF MS 分析鉴定蚊子的最佳条件。
我们使用三种均化方法（两种手动和一种自动）对两种实验室蚊子品系（Anopheles coluzzii 和 Aedes aegypti）的三个不同身体部位（腿、胸、头）进行了处理，并对结果进行了评估。MS 图谱的重现性、相关评分的鉴定率以及高通量分析的适用性是建立优化指南的主要标准。此外，还使用实验室品系和现场采集的蚊子评估了血液摄入和地理来源的影响。
使用 MALDI-TOF MS 分析对所有三个部位进行蚊子鉴定都获得了相关评分值；然而，无论均化方法或物种如何，胸部和腿部都是最适合的标本。虽然手动均化方法与三个部位的高鉴定率相关，但这种均化模式不适用于大量样本的处理。因此，选择自动均化程序作为参考均化方法。尽管在两种物种的三个部位测试的 MS 图谱中都存在原始血液的 MS 峰，但血液摄入状态并没有阻碍蚊子物种的鉴定。最后，当向数据库中添加来自同一地理区域的物种的 MS 谱时，从同一地理区域采集的标本的鉴定得分得到了显著提高。
本研究结果为 MALDI-TOF MS 图谱鉴定未来标本的蚊子解剖部位选择和样品制备方式（如均化）建立了指南。这些标准化操作协议可以作为创建国际 MS 数据库的参考。
