Development of liquid AP-MALDI mass spectrometry (MS) for microbial profiling and biotyping

液相AP-MALDI质谱（MS）用于微生物谱分析和生物分型的研究利用单电荷肽离子的MALDI质谱谱进行微生物生物分型，是一种比传统的临床微生物鉴定方法上级的方法，大大提高了微生物的分类精度和分析速度。最近，这导致了两个FDA批准的系统，用于通过MALDI MS生物分型进行微生物检测和鉴定。该方法目前正在进一步开发中，以便将其扩展到AMR检测。我们建议通过使用我们新开发的基于激光的AP-MALDI离子源，利用其独特的能力，（肽和蛋白质）分析物离子以及通过使用液体样品在样品制备中的更大速度和灵活性以及本质上更简单的离子源设计。由于多电荷离子的众所周知的上级碎裂作用，通过我们的新方法对多电荷离子的鉴定将允许通过MS/MS测序进行上级鉴定。它还将允许通过离子迁移率过滤和使用高性能质谱仪如轨道捕获器来有效消除单电荷生物基质背景离子（例如来自培养基）。这些优点以及更简单的离子源设计的优点，从而提高了样品访问速度，是目前FDA批准的MALDI-TOF MS生物分型技术无法实现的。因此，拟议的博士生项目将研究（A）通过利用多电荷MALDI离子的MS/MS测序进行上级微生物鉴定，（B）使用离子迁移率过滤使信噪比增加大于或等于100，（C）液体基质组合物的更大灵活性和它们在为AMR检测测定提供足够环境方面的潜力，和（D）由于使用了大气压（AP）离子源和更简单的样品制备，提高了分析速度。组装的监督团队由分析化学、液体和（临床）微生物学，连接阅读大学和皇家伯克希尔医院，在H&S和其他必要的诱导/评估之后，该项目的研究部分将从测试和优化微生物培养物的液体MALDI MS分析的提取方案开始，最初使用的是从吉布森教授的研究小组获得的乳酸菌。这些细菌有几个优点：它们在大学里得到了很好的表征和理解，有一个测序的基因组，并且可以安全地处理。在本初步工作中，将测试方法的最短培养时间和提取，以提供足够量的特定标记离子，可用于通过MS/MS测序进行明确鉴定（见目标A）。上述工作将通过使用离子迁移率过滤进行扩展，这将进一步提高信噪比（见目标B）。在这里，将研究不同的过滤器设置，例如用于单电荷离子的消除过滤器的各种组合和用于特定类别的生物分子的特定过滤器窗口（例如肽与蛋白质与脂质）。此外，还将研究临床分离株的提取方案，以实现最佳提取并提供临床菌株的生物安全提取物。接下来，将开发AMR检测方法，该方法将利用微生物内酰胺酶活性水解抗生素的β-内酰胺环后容易检测的质量变化。在此，将确定酶、底物和缓冲液的最佳添加，以实现有效的底物回收和随后的MS分析（参见目标C）。

Development of liquid AP-MALDI mass spectrometry (MS) for microbial profiling and biotypingMicrobial biotyping using MALDI mass spectral profiles of singly charged peptide ions has been recently established as a superior method to classical clinical microbiology assays for the identification of clinically relevant microbes with substantially increased classification accuracy and speed of analysis. Recently, this has led to two FDA-approved systems for microbial detection and identification by MALDI MS biotyping. This methodology is now under further development for extending it to AMR detection.We propose to advance this methodology even further by using our newly developed laser-based AP-MALDI ion source, exploiting its unique capability to create multiply charged (peptide and protein) analyte ions as well as its greater speed and flexibility in sample preparation through the use of liquid samples and essentially a simpler ion source design.The availability of multiply charged ions through our new methodology will allow superior identification by MS/MS sequencing due to the well-known superior fragmentation of multiply charged ions. It will also allow the efficient elimination of singly charged biological matrix background ions (e.g. from the culture media) by ion mobility filtering and the use of high-performing mass spectrometers such as orbitraps. These and the advantages of a simpler ion source design, and thus increased sample access speed, are not achievable with the current FDA-approved MALDI-TOF MS technologies for biotyping.Thus, the proposed PhD studentship will investigate the (A) superior microbial identification by utilising MS/MS sequencing of multiply charged MALDI ions, (B) increase in the signal-to-noise ratio by greater than or equal to 100 using ion mobility filtering, (C) greater flexibility in liquid matrix compositions and their potential in providing an adequate environment for AMR detection assays, and (D) increase in analytical speed due to the use of an atmospheric pressure (AP) ion source and simpler sample preparations.The assembled supervisory team consists of experts in the field of analytical chemistry, liquids and (clinical) microbiology, linking the University of Reading with the Royal Berkshire Hospital, and includes scientist at different stages of their academic careers.WorkplanAfter H&S and other essential inductions/assessments, the research part of the project will start with testing and optimising extraction protocols for liquid MALDI MS analysis of microbial cultures, initially using lactobacteria, which will be obtained from Prof Gibson's group. These bacteria have several advantages: they are well-characterised and understood here at the University, have a sequenced genome and are safe to handle. In this preliminary work, methods will be tested for shortest culture times and extractions that provide sufficient amounts of specific marker ions that can be used for unambiguous identification by MS/MS sequencing (see objective A).The above work will be extended by using ion mobility filtering, which will further improve the signal-to-noise ratio (see objective B). Here, different filter settings will be investigated such as various combinations of elimination filters for single charged ions and specific filter windows for specific classes of biomolecules (e.g. peptides vs proteins vs lipids). In addition, extraction protocols for clinical isolates will be investigated with the goal to achieve both best extraction and the provision of biologically safe extracts from clinical bacterial strains.Next, AMR assays will be developed that will take advantage of the easily detectable mass shift after hydrolysis of the beta-lactam rings of antibiotics by microbial lactamase activity. Here, the optimal addition of enzyme, substrate and buffers will be determined with a view on efficient substrate recovery and subsequent MS analysis (see objective C).