Uhlmann Katrin R, Gibb Sebastian, Kalkhof Stefan, Arroyo-Abad Uriel, Schulz Claudia, Hoffmann Bernd, Stubbins Francesca, Carpenter Simon, Beer Martin, von Bergen Martin, Feltens Ralph
Department of Proteomics, Helmholtz-Centre for Environmental Research-UFZ, 04318 Leipzig, Germany.
Parasit Vectors. 2014 Aug 24;7:392. doi: 10.1186/1756-3305-7-392.
Culicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides.
Proteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract.
We showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data.
Shotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.
库蠓是蓝舌病病毒和施马伦贝格病毒的传播媒介，这些病毒在欧洲的反刍家畜中引发大规模疾病流行。基于形态特征和基因标记测序的方法最常用于将库蠓区分到物种水平。然而，蛋白质组学方法也越来越多地被用作一种替代鉴定方法。这些技术有可能快速，并且可能比基于DNA的技术具有优势。这项原理验证研究的目的是开发一种基于基质辅助激光解吸/电离飞行时间质谱（MALDI-MS）的简单方法，通过肽模式区分不同物种的库蠓，并额外选择鉴定具有鉴别性的肽。
从7种库蠓物种中提取的蛋白质进行消化，并对所得肽进行纯化。使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）记录肽质量指纹（PMF）光谱，并使用MALDIquant R软件包在R中分析峰模式。此外，应用离线液相色谱（LC）MALDI-TOF串联质谱（MS/MS）来确定使用未分级提取物获得的一个示例性MALDI光谱中肽峰的身份。
我们表明，大多数库蠓物种产生了具有适合分类的峰模式的可重复质谱。通过质谱获得的树状图与由细胞色素氧化酶I（COX1）序列生成的树状图显示出初步的相似性。使用离线LC-MALDI-TOF-MS/MS，我们确定了在一个MALDI光谱中观察到的28个肽峰的身份，质量范围为1.1至3.1 kDa。由于缺乏可用的库蠓数据，所有鉴定出的肽与其他双翅目物种相同，并且源自五种高度丰富的蛋白质之一。
已证明通过MALDI-TOF-MS进行鸟枪法质量图谱分析与标本的形态学和基因鉴定兼容。此外，该方法的性能至少与基于完整蛋白质质谱的替代方法一样好，从而将该程序确立为一种独立的方法，并且额外选择了同时在其他基于质谱的蛋白质鉴定应用中使用相同的样品。不同库蠓物种的基因组信息的未来可用性可能使基于库蠓特异性序列信息的更严格的肽检测成为可能。
