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

开发用于微生物分析和生物分型的液体 AP-MALDI 质谱 (MS)

• 批准号: 2104529
• 金额: --
• 依托单位: University of Reading
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2104529
• 关键词: Development; liquid; AP; MALDI; mass

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).

用于微生物分析和生物分型的液体 AP-MALDI 质谱 (MS) 的开发 最近，使用单电荷肽离子的 MALDI 质谱图谱进行微生物生物分型已被确立为优于传统临床微生物学检测的方法，用于鉴定临床相关微生物，并且大大提高了分类准确性和分析速度。最近，这导致了 FDA 批准的两个通过 MALDI MS 生物分型进行微生物检测和识别的系统。该方法目前正在进一步开发，以将其扩展到 AMR 检测。我们建议通过使用我们新开发的基于激光的 AP-MALDI 离子源进一步推进该方法，利用其独特的能力来产生多电荷（肽和蛋白质）分析物离子，以及通过使用液体样品和更简单的离子源设计在样品制备中提高速度和灵活性。通过我们的新方法获得多电荷离子将 由于众所周知的多电荷离子卓越的碎裂能力，可以通过 MS/MS 测序进行卓越的鉴定。它还可以通过离子淌度过滤和使用高性能质谱仪（例如轨道阱）有效消除单电荷生物基质背景离子（例如来自培养基）。目前 FDA 批准的用于生物分型的 MALDI-TOF MS 技术无法实现这些优点以及更简单的离子源设计，从而提高样品获取速度。因此，拟议的博士生奖学金将研究 (A) 通过利用多电荷 MALDI 离子的 MS/MS 测序实现卓越的微生物鉴定，(B) 使用离子将信噪比提高大于或等于 100 迁移率过滤，(C) 液体基质成分的更大灵活性及其为 AMR 检测分析提供适当环境的潜力，以及 (D) 由于使用大气压 (AP) 离子源和更简单的样品制备而提高了分析速度。组建的监督团队由分析化学、液体和（临床）微生物学领域的专家组成，将雷丁大学与皇家伯克希尔医院联系在一起， 工作计划在 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液体 MALDI MS 分析的提取方案开始，最初使用从 Gibson 教授小组获得的乳酸菌。这些细菌有几个优点：它们在大学里得到了很好的表征和了解，具有经过测序的基因组并且可以安全处理。在这项初步工作中，将测试最短培养时间和提取的方法，以提供足够量的特定标记离子，可用于通过 MS/MS 测序进行明确识别（参见目标 A）。上述工作将通过使用离子淌度过滤进行扩展，这将进一步提高信噪比（参见目标 B）。在这里，将研究不同的过滤器设置，例如用于单带电离子的消除过滤器的各种组合和用于特定类别生物分子（例如肽、蛋白质、脂质）的特定过滤器窗口。此外，还将研究临床分离株的提取方案，目标是实现最佳提取并从临床细菌菌株中提供生物安全的提取物。下一步，将开发 AMR 测定法，利用微生物内酰胺酶活性水解抗生素 β-内酰胺环后容易检测到的质量转移。在这里，酶、底物和缓冲液的最佳添加量将根据底物的有效回收和随后的 MS 分析来确定（参见目标 C）。