A Cost-Effective High-Speed Clinical Diagnostics Instrument for Large Population Screening Based on Novel Liquid AP-MALDI MS Technology
Lead Research Organisation:
University of Reading
Department Name: Chemistry
Abstract
Based on previously funded EPSRC research (EP/L006227/1) for the 'Development of a novel MALDI mass spectrometer and technology for the generation of multiply charged ions at high sensitivity' and subsequent initial exploitation of this new technology, the proposed project will develop a new instrument that specifically fulfils key requirements in clinical diagnostics as demanded by modern medicine, in particular in the age of new pandemics such as COVID-19.
Accurate and fast characterisation of microorganisms in clinical samples are crucial for initiating optimal treatment and limiting the outbreaks of pandemics. Both accuracy and time are key to the best treatment outcome for the patient, minimising the time to recovery and more importantly minimising morbidity and mortality. In particular, the correct and rapid identification of newly discovered microbial pathogens or antimicrobial-resistant strains is important for the patient's recovery. Combined with the capability of large-scale testing, it will also allow for a better global response to (microbial) infectious diseases.
Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) profiling of organisms (biotyping) has recently been 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. This has already led to two FDA approved systems for microbial detection and identification by MALDI MS biotyping. In the proposed project, this methodology will be substantially advanced by exploiting multiply charged ions and their co-analysis with lipids and other biomolecules on a bench-top MS/MS instrument specifically optimised for large-scale, inexpensive clinical analyses, thus leading to the next generation of superior MALDI MS biotyping for clinical use and mass testing.
The unique features of this new instrument and the associated technology will be high speed, cost-effectiveness, and high specificity by MS/MS sequencing. Combined with the unrivalled resolution of mass spectrometry this new technology will be a step-change in diagnostic testing by allowing the testing of multiple diseases within the same test run as well as being highly adaptable to new diseases without the need to develop test reagents that are disease/microbe-specific, difficult to source and therefore expensive, in particular for newly discovered diseases (cf. COVID 19). The aim is to reach a throughput level of 100,000 samples per day at high detection accuracy and low cost per sample.
From collaborating with the biopharmaceutical industry and analytical instrument manufacturers, and from research of a BBSRC-funded grant, we found that our novel AP-MALDI MS ion source provides a sensitive platform for rapid assay analysis with the potential for use in the early detection of microbial diseases. The proposed project will build on this preliminary data, develop a new tailor-made instrument for future clinical use and explore the advantages (compared to current MALDI MS biotypers) in (a) speed, (b) the elimination of biological matrix background, (c) superior MS/MS analysis, (d) greater ion signal stability, (e) multiplexing capability and (f) the simpler (and cost-effective) but more flexible sample preparation that this new technology can offer.
Accurate and fast characterisation of microorganisms in clinical samples are crucial for initiating optimal treatment and limiting the outbreaks of pandemics. Both accuracy and time are key to the best treatment outcome for the patient, minimising the time to recovery and more importantly minimising morbidity and mortality. In particular, the correct and rapid identification of newly discovered microbial pathogens or antimicrobial-resistant strains is important for the patient's recovery. Combined with the capability of large-scale testing, it will also allow for a better global response to (microbial) infectious diseases.
Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) profiling of organisms (biotyping) has recently been 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. This has already led to two FDA approved systems for microbial detection and identification by MALDI MS biotyping. In the proposed project, this methodology will be substantially advanced by exploiting multiply charged ions and their co-analysis with lipids and other biomolecules on a bench-top MS/MS instrument specifically optimised for large-scale, inexpensive clinical analyses, thus leading to the next generation of superior MALDI MS biotyping for clinical use and mass testing.
The unique features of this new instrument and the associated technology will be high speed, cost-effectiveness, and high specificity by MS/MS sequencing. Combined with the unrivalled resolution of mass spectrometry this new technology will be a step-change in diagnostic testing by allowing the testing of multiple diseases within the same test run as well as being highly adaptable to new diseases without the need to develop test reagents that are disease/microbe-specific, difficult to source and therefore expensive, in particular for newly discovered diseases (cf. COVID 19). The aim is to reach a throughput level of 100,000 samples per day at high detection accuracy and low cost per sample.
From collaborating with the biopharmaceutical industry and analytical instrument manufacturers, and from research of a BBSRC-funded grant, we found that our novel AP-MALDI MS ion source provides a sensitive platform for rapid assay analysis with the potential for use in the early detection of microbial diseases. The proposed project will build on this preliminary data, develop a new tailor-made instrument for future clinical use and explore the advantages (compared to current MALDI MS biotypers) in (a) speed, (b) the elimination of biological matrix background, (c) superior MS/MS analysis, (d) greater ion signal stability, (e) multiplexing capability and (f) the simpler (and cost-effective) but more flexible sample preparation that this new technology can offer.
People |
ORCID iD |
Rainer Cramer (Principal Investigator) | |
Ian Jones (Co-Investigator) |
Publications
![publication icon](/resources/img/placeholder-60x60.png)
Adair LR
(2024)
Utilizing Precursor Ion Connectivity of Different Charge States to Improve Peptide and Protein Identification in MS/MS Analysis.
in Analytical chemistry
![publication icon](/resources/img/placeholder-60x60.png)
Challen B
(2022)
Advances in ionisation techniques for mass spectrometry-based omics research
in PROTEOMICS
![publication icon](/resources/img/placeholder-60x60.png)
Challen B
(2023)
Ultra-High-Throughput and Low-Volume Analysis of Intact Proteins with LAP-MALDI MS.
in Journal of the American Society for Mass Spectrometry
![publication icon](/resources/img/placeholder-60x60.png)
Krenkel H
(2023)
The use of salts, buffers and surfactants in LAP-MALDI MS
in International Journal of Mass Spectrometry
Description | Improvements to our new mass spectrometry technology LAP-MALDI, leading to higher sensitivity |
Exploitation Route | A patent application has been filed by the University of Reading |
Sectors | Other |
Description | BBSRC International Partnerships |
Amount | £270,830 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2024 |
End | 06/2024 |
Description | HIP - IMPROV |
Amount | £8,000 (GBP) |
Funding ID | IMPROV |
Organisation | University of Reading |
Sector | Academic/University |
Country | United Kingdom |
Start | 08/2022 |
Description | Health Innovation Partnership funding for biofluid sample collection from NHS patients |
Amount | £23,593 (GBP) |
Organisation | University of Reading |
Sector | Academic/University |
Country | United Kingdom |
Start | 03/2024 |
End | 03/2025 |
Description | PhD strategic studentship: Novel applications of liquid AP-MALDI MS |
Amount | £80,000 (GBP) |
Funding ID | GS23-015 |
Organisation | University of Reading |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2023 |
End | 09/2026 |
Title | A new benchtop Q-TOF mass spectrometer with LAP-MALDI ion source |
Description | This new benchtop Q-TOF mass spectrometer is equipped with a LAP-MALDI ion source which allows mass spectrometry measurements using the liquid atmospheric pressure MALDI MS technique. This setup is comparable to the design of a previously developed ion source (see research grant EP/L006227/1) with slight geometric modifications to have a more compact design for benchtop mass spectrometers and is the first benchtop mass spectrometer with a LAP-MALDI ion source. |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | This setup allows mass spectrometry measurements of various types of sample in a high-throughput manner, reaching subsecond cycle times per sample with all of the advantages of LAP-MALDI MS such as the generation of multiply charged ions, contactless sampling, highly stable energy deposition and great sample homogeneity (and therefore highly stable ion yields). This LAP-MALDI mass spectrometer setup, which is much more compact in its design than previous setups, might serve as a blueprint for future developments of this technology towards commercialised versions applying this technology. |
Title | Liquid Biopsies from Royal Berkshire Hospital Participants |
Description | Pathogen identification in clinical samples by mass spectrometry is predominantly performed using samples that are obtained after several hours or days of cultivation and then prepared to result in solid samples. In this study, we aim to test an alternative using liquid samples on an instrument, providing additional functionalities and ultimately with superior mass spectrometric performance. This method (liquid AP-MALDI MS) has shown good potential in outperforming current methods in both analysis speed and specificity. Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid and protein profiles. the method will be tested in liquid biopsy samples collected from Royal Berkshire NHS Foundation Trust (RBFT) volunteers/participants. The Health Research Authority and the University of Reading Ethics Committee have approved the sample collection in Dec 2022. A small number of different types of samples such as saliva, sebum, finger-prick blood and nasal swabs are being collected from consenting participants. Samples are being collected based on standard operating procedures (SOPs) and processed in the University of Reading Class II analytical lab. For the instrument optimisation and methodology development, different sample preparation (two-step method) techniques are being developed such as TCA and TFA precipitation for protein and metabolite extraction. Biological samples such as saliva or bacterial cultures are being used for the method development. After the development of extraction procedure, the processed samples will be tested using ion-exchange beads, to explore whether abundant sodium adduct can be removed from the extracted samples. The best method will be then adapted for the analysis of liquid biopsy samples collected from RBFT participants. |
Type Of Material | Biological samples |
Year Produced | 2024 |
Provided To Others? | No |
Impact | The research method development is ongoing. |
Title | Modifications for (LAP-)MALDI ion sources to raise sensitivity of the (LAP-)MALDI MS method |
Description | Experimental works have been conducted on the earlier reported LAP-MALDI MS source, which is coupled to the Xevo G2-XS Q-Tof mass spectrometer. The purpose of these modifications are to improve the overall sensitivity of the ion source. It was understood from preliminary experiments with peptide-spiked human biofluid samples, that the sensitivity of the instrumental setup was the most crucial factor, apart from proper sample preparation, to detect reliably biomarkers of interest for the diseases from biofluid samples, including those collected from patients. The modifications include the introduction of optical elements along the ion beam pathway such as beam expanders and holographic lenses. Moreover, 3D-printed devices mounted on the ion transfer capillary are used to enhance ion transmission. |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2023 |
Provided To Others? | No |
Impact | Each of these modifications added a moderate enhancement in sensitivity for peptide analytes to a factor of around 2x. The next step will be a combination of all these modifications to evaluate if the sensitivity can be further raised when these tools are used in concert. |
Title | Reduction of alkaline ion formation in MS to improve LAP-MALDI MS sensitivity through the use of ion exchange beads |
Description | Alkaline adduct formation in LAP-MALDI MS particularly from Na+/K+ ions, a phenomenon which is also often observed for electrospray ionization, usually leads to protonated ion suppression and ion signal dilution, and thus ultimately leads to a lowering of the limit of detection of the analyte(s) of interest. Biological fluids, due to their nature, contain high concentration of alkaline ions. We have introduced the use of different mesh-sized and pre-washed, loaded ion exchange beads (AmberLite HPR650 H, AmberLite MB, Dowex 50WX8) which have sulfonic acid functional groups as ion exchange moieties, to be inserted into the liquid samples for the reduction of alkaline adduct formation. |
Type Of Material | Technology assay or reagent |
Year Produced | 2023 |
Provided To Others? | No |
Impact | The method included either the controlled insertion of a number of beads (e.g. 1 - 3, depending on the mesh size) directly to the sample droplets on the MALDI target prior analysis. Within the alternative workflow, beads were transferred to the (peptide) sample for a pre-mix step (vortexing). Then, a sample aliquot (without beads) from the supernatant was taken for analysis. Currently, the method is being further refined to have a more defined transfer of such beads to the samples and to select the most effective ion exchange bead type. It is planned to establish these beads as an integral part of the biological sample preparation procedure. |
Title | Dataset to support improved peptide and protein database search scores in chimeric MS/MS spectra |
Description | This dataset contains raw and processed data for liquid atmospheric-pressure matrix-assisted laser desorption/ionisation (LAP-MALDI) mass spectrometry analysis of mixtures of peptides and proteins using a modified Synapt G2-Si with an in-house built atmospheric pressure MALDI source. Chimeric MS/MS peak lists and filtered peak lists produced through the presented data acquisition/analysis workflow were searched using MASCOT. |
Type Of Material | Database/Collection of data |
Year Produced | 2023 |
Provided To Others? | Yes |
URL | https://researchdata.reading.ac.uk/id/eprint/476 |
Description | RBH HIP collaboration |
Organisation | Royal Berkshire NHS Foundation Trust |
Country | United Kingdom |
Sector | Public |
PI Contribution | We have supported biological sample collection by obtaining HRA and UREC ethical approval for research in new disease diagnostics methods development using LAP-MALDI MS. |
Collaborator Contribution | RBH consultants have supported this project with their time and expert knowledge in infectious disease diagnostics. |
Impact | Clinical sample collection of biofluids from NHS patients. |
Start Year | 2021 |
Description | Waters Corporation |
Organisation | Waters Corporation |
Department | Micromass UK Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | Development of new ion source |
Collaborator Contribution | Support and provision of instrument details for the above |
Impact | Additional funding for studentships |
Start Year | 2012 |
Title | DEVICE AND METHOD FOR AEROSOL COLLECTION |
Description | The present invention relates to a device and method for collecting aerosols produced by ablation, desorption or evaporation techniques. Specifically, the aerosols may be collected for use in analytical instrumentation such as a mass spectrometer. |
IP Reference | 2310773.3 |
Protection | Patent / Patent application |
Year Protection Granted | 2023 |
Licensed | Commercial In Confidence |
Impact | The present invention improves the ion signal (sensitivity) by a factor of 2 or more on mass spectrometers with an AP-MALDI ion source. Its IP is currently being considered in the context of our commercialisation efforts. |
Description | Website related to EPSRC award EP/V047485/1 |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | A word-press based website supported by the University of Reading and titled with "Fast speed diagnostics" was created describing the above EPSRC project and its project outcomes in order to inform on the research topic. Website link: https://research.reading.ac.uk/highspeeddiagnostics Participants providing samples for this project are referred to this website for obtaining further information. |
Year(s) Of Engagement Activity | 2022 |
URL | https://research.reading.ac.uk/highspeeddiagnostics |