Development of a Novel MALDI Mass Spectrometer and Technology for the Generation of Multiply Charged Ions at High Sensitivity

Lead Research Organisation: University of Reading
Department Name: Chemistry

Abstract

Modern mass spectrometry (MS) can be compared to microscopy with its impact on analysing nature and materials down to the molecular (individual molecule) level, including the analysis of the cellular processes of life and the structure of molecules. These analytical tools have frequently been the key to major breakthroughs in science. MS in particular, has been at the forefront of recent advances in areas like biomedicine and healthcare. In this project we will develop a new instrument and the associated methodologies for another major step forward in MS and its application.

MS requires the production of gas phase ions. The two major ionisation techniques in modern MS are electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI). There are fundamental differences between these two techniques. ESI enables ion formation exclusively out of a liquid while MALDI uses predominantly solid samples. Another significant difference can be found in their ability to produce multiply charged ions. For peptides, MALDI typically generates singly charged while ESI easily provides multiply charged ions.

Importantly, the production of highly charged ions is desirable as it allows the use of high-performance mass spectrometers, which typically cannot analyse the larger singly charged ions. It also facilitates more informative controlled fragmentation of the ions, thus helping to obtain further information such as their molecular structure. Consequently, there is a clear advantage of using ESI. Nonetheless, MALDI with its higher tolerance to contaminants, ease-of-operation, potential for high-speed automated analysis as well as its MS imaging capabilities makes it an ionisation technique that can cover (bio)analytical areas where ESI is less suitable. If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and new large-scale (bio)analyses using MALDI MS would become feasible.

The proposed instrument and method development will lead to a new technology that will enable the production of stable and high yields of multiply charged MALDI ions at high sensitivity, i.e. low analyte concentration and low sample consumption. It is based on a new ion source design, using a heated ion transfer tube to transfer the produced ions into the analyser of the mass spectrometer, and novel liquid sample preparation methods, ensuring stable and high yields of ESI-like multiply charged ions. Thus, the two main disadvantages of MALDI (no/low yield of multiply charged ions and highly variable ion yield and signal quality) will be addressed within this project. Ultimately, the newly developed technology should not only become a real competitor for ESI but also open up new areas of analysis that have previously been inaccessible.

In short, this project will develop a new MS technology that will significantly widen the application range of MALDI MS and thus MS in general, enabling new and more powerful analytical strategies. The project will result in a prototype instrument and methodology that can easily be commercialised. As MALDI MS is already making great strides within the (bio)analytical field it can be anticipated that this project will have significant impact in many areas from academia and industry to the public health sector and thus society at large.

Planned Impact

Novel and improved analytical tools have frequently been the key to major breakthroughs in science.
Mass spectrometry (MS) in particular, has been at the forefront of recent advances in areas like biomedicine and healthcare. The MALDI mass spectrometer and technology that will be developed in this project will significantly widen the application range of MALDI, enabling new and more powerful analytical strategies. As MALDI MS is already making great strides within the bioanalytical arena it can be anticipated that this project will have significant impact in many areas from academia and industry to the public health sector and society at large.

Who might benefit from this research?

- Academia: MS researchers and all researchers applying MS as an analytical tool, from physicists and chemists to biomedical and clinical scientists. As MS is employed in molecular analysis, post-genomic research and increasingly in clinical research, this new technology has the potential to accelerate the work of an enormously wide research community.
How might they benefit? MS is often compared to microscopy. Similarly to microscopy that has enabled us to 'look' at cellular processes and molecular structure and interaction, MS is one of the modern analytical techniques that has the sensitivity and capability to reveal and investigate molecular processes further, from synthetic chemistry to systems biology and medicine. This new MALDI-based technique can combine the advantages of the competitor technique ESI (electrospray ionisation) with the advantages of MALDI and overcome some of the restrictive disadvantages of ESI, making it a technique that will be immensely powerful in modern MS. One project outcome will be the implementation of this new technology, resulting in a semi-commercial instrument. Thus, this project will provide the means and evidence for the technology's direct impact on studies and set-ups using modern mass spectrometers.

- Business/Industry: MS manufacturers and industry applying modern MS in research and analysis.
How might they benefit? New innovations in MS have been a major driver for the analytical sciences industry. MS is a multi billion dollar business and thus new technology in this field will have a major impact on the manufacturing industry, particular if it is a fundamental advance. In industry where MS is applied this new technology has the potential to enable the analysis of new substance classes under conditions previously impossible to employ. In combination with its speed and automation capabilities, major steps towards large-scale and rapid analysis in important areas such as diagnostics and screening can be foreseen, potentially leading to the next step for MS to be firmly established in areas such as healthcare (clinical labs), food security/testing, and other environmental analyses where modern MS is required.

- General Public: Various sections of the world population (e.g. patients)
How might they benefit? In general, there is great potential for advances in areas where organic macromolecules and their analysis play a major role, from the polymer sciences (new materials) to biomedicine, and society at large will benefit from these advances through better living conditions, economic advances and health, to name but a few. One specific example is the healthcare system where patients will indirectly benefit through new research and data in the life and biomedical sciences as well as through new diagnostic capabilities, all made possible with improved analytical methods such as the presented one.

- Public/Third Sector/Policy Makers/Governments: Research sponsors/funders, Science Advisors, (public) analytical laboratories
How might they benefit? Through new possibilities in funding research and savings from employing this new technology and its capabilities in providing deeper and more effective analyses.

Publications

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Cramer R (2016) 'Next generation' laser-based biological mass spectrometry. in Methods (San Diego, Calif.)

 
Description We have developed a new analytical tool and found new optimization strategies for laser-based ionization in maximizing ion yields of multiply charged analyte ions, including large biomolecules such as proteins. These can now be analysed on high-performing mass spectrometers, previously inaccessible for such molecules using laser-based ionization techniques.
All this will help to inform new hardware developments in mass spectrometry and its application in the life sciences.
Exploitation Route Our findings can be taken forward by industry for developing new laser-based mass spectrometers, significantly expanding the breadth of mass spectrometry analysis. For instance, the University of Reading has recently assigned relevant IP as published under WO2014096847 to Micromass UK Ltd. (EP13815814). The findings can also lead to new applications of MALDI MS in the life sciences such as food science and agriculture as can be seen by recent research grants awarded in these areas.
Sectors Agriculture, Food and Drink,Other

 
Description Our findings are also being explored by industry. The University of Reading has assigned relevant IP as published under WO2014096847 to Micromass UK Ltd. (EP13815814). Further collaboration with Waters Corporation is underway. In 2017/2018, further interest has been recorded from industry overseas. This has partially led to an industrially funded research project and is also being explored for further industrial applications.
First Year Of Impact 2013
Sector Agriculture, Food and Drink,Other
Impact Types Economic

 
Description DAAD RISE Worldwide
Amount € 5,000 (EUR)
Organisation German Academic Exchange Service (DAAD) 
Sector Academic/University
Country United States
Start 07/2017 
End 10/2017
 
Description DAAD RISE Worldwide
Amount € 5,000 (EUR)
Organisation German Academic Exchange Service (DAAD) 
Sector Academic/University
Country United States
Start 07/2018 
End 10/2018
 
Description EPSRC DTG studentship with industrial contribution
Amount £34,250 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 03/2017
 
Description EPSRC DTG studentship with industrial contribution
Amount £35,000 (GBP)
Organisation Waters Corporation 
Sector Private
Country United States
Start 10/2014 
End 03/2018
 
Description EPSRC DTP studentship with industrial contribution
Amount £35,000 (GBP)
Organisation Waters Corporation 
Sector Private
Country United States
Start 09/2016 
End 09/2019
 
Description EPSRC DTP studentship with industrial contribution
Amount £30,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 09/2019
 
Description Equipment loan for HTS by AP-MALDI MS
Amount £20,000 (GBP)
Organisation Waters Corporation 
Sector Private
Country United States
Start 11/2019 
 
Description Peptidomic analysis of the protein fraction of Stablor® before and after bacterial fermentation
Amount £146,858 (GBP)
Organisation LNC Therapeutics 
Sector Private
Country France
Start 01/2018 
End 12/2018
 
Description UoR-funded studentship
Amount £30,000 (GBP)
Organisation University of Reading 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 09/2021
 
Description Waters-funded PhD studentship
Amount £33,000 (GBP)
Organisation Waters Corporation 
Sector Private
Country United States
Start 10/2018 
End 09/2021
 
Title Detection of clinical and preclinical mastitis 
Description Introduction: Liquid AP-MALDI as a modified ion source were used to analyse homogenous liquid sample droplets on a commercial high-performing Q-TOF mass spectrometer with ion mobility separation. This system has the advantage of being able to produce and analyze MALDI-generated protein ions with high charge states. As the arrangement allows for the detection of high-mass proteoforms in an m/z range below 2000, the full power of modern hybrid mass analyzers can be utililzed for MALDI, in the same way as for ESI-based analyses. Thus, it is possible to detect diagnostically relevant proteins, lipids and metabolites in the same MALDI-MS profile and achieve structural analysis of these by high-quality MS/MS and from the same sample by just performing a simple, one-pot sample preparation. Methods: A milk biobank of 12,000 samples was established by collecting weekly samples from around 500 cows at the CEDAR research farm at the University of Reading, UK. Milk aliquots from this biobank (20µL) and from milk bought at local groceries (50µL) were precipitated with trichloroacetic acid and re-suspended in water/acetonitrile/isopropanol. The analyte extraction solution (0.65uL) was added to a pre-spotted liquid support matrix (LSM) droplet (0.65uL; CHCA 30 mg/mL in acetonitrile:water, 70:30 (v/v), mixed with ethylene glycol in a ratio of 10:7). Automated MS data acquisition was achieved with a Waters Synapt G2-Si and an in-house developed AP-MALDI ion source. Data analysis was undertaken with AMX (Waters) software. Preliminary data: Using milk bought at local groceries, goat milk adulterated with 5% cow milk was detected with 92.45% sensitivity and 94.53% specificity. Goat milk adulterated with 10% cow milk was detected with 99.15% sensitivity and 99.10% specificity. Using aliquots from the milk biobank, clinical bovine mastitis (n=60) was detected with a classification accuray of 98.52% using a large control group (n=327) that included a subgroup with high somatic cell counts (hSCC, n=106). Pre-clinical mastitis (PCM) was detected with 85% correct classification rate for up to four days before the clinical mastitis event and with 95% correct classification rate two days before the clinical mastitis event. These results demonstrate the potential of liquid AP-MALDI MS profiling coupled with multivariate analysis for robust and highly accurate profiling using lipid and protein MS profiles in biological specimens. This was achieved with stable ion yields and low sample consumption. Its simplicity and applicability to high-performing hybrid MS instrumentation manufactured for the use of AP ion sources makes it a MALDI MS method that can uniquely take advantage of the performance of such instrumentation, including speed in changing samples and various analysis modes in a very short time. Examples of MS/MS structural analysis of relevant biomarkers from the same liquid MALDI sample will also be presented. Novel aspect: High-throughput AP-MALDI MS as a novel biotyping approach combining high-sensitivity detection and MS/MS analysis of ESI-like multiply charged analytes. Oral Sessions: Food Safety & Chemistry: Innovations; Food Safety & Chemistry: Foodomics, Allergens, Bacteria, Foods, and Supplements Poster Sessions: Food Safety: General; Ambient Ionization: Applications; MALDI: Applications 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact These results demonstrate the potential of liquid AP-MALDI MS profiling coupled with multivariate analysis for robust and highly accurate profiling using lipid and protein MS profiles in biological specimens. This was achieved with stable ion yields and low sample consumption. Its simplicity and applicability to high-performing hybrid MS instrumentation manufactured for the use of AP ion sources makes it a MALDI MS method that can uniquely take advantage of the performance of such instrumentation, including speed in changing samples and various analysis modes in a very short time. Examples of MS/MS structural analysis of relevant biomarkers from the same liquid MALDI sample will also be presented. 
 
Title New AP-MALDI MS ion source 
Description A new mass spectrometry ion source capable of generating multiply charged MALDI ions at high sensitivity. 
Type Of Material Improvements to research infrastructure 
Year Produced 2013 
Provided To Others? Yes  
Impact This new invention resulted in several lines of exploitation for the application of MALDI in classical modern MS analysis fields. Our industrial collaborator Waters has also taken this forward by further investigating its future use. 
 
Title New AP-MALDI MS ion source 
Description A new mass spectrometry ion source capable of generating multiply charged MALDI ions at high sensitivity. 
Type Of Material Improvements to research infrastructure 
Year Produced 2013 
Provided To Others? Yes  
Impact This new invention resulted in several lines of exploitation for the application of MALDI in classical modern MS analysis fields. Our industrial collaborator Waters has also taken this forward by further investigating its future use. 
 
Title Dataset supporting research into the production and analysis of multiply charged negative ions by atmospheric pressure matrix-assisted laser desorption/ionisation mass spectrometry 
Description The dataset contains mass spectrometry data which was used as the basis for a published article focusing on the development of our in-house AP-MALDI ion source for negative ion production and analysis. The data is in the proprietary Waters .raw format, but can be converted to be viewed in free and open source software. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
 
Title Heatmap script 
Description Script to post process and visualise collected MS data to find optimal operational parameters 
Type Of Material Data analysis technique 
Provided To Others? No  
Impact Allowed to visualise large subset of data and helped to analyze and get better understanding of the ionization mechanisms. Allowed to fine tune experimental set-up 
 
Description Analysis of blood samples for forensic investigation 
Organisation Sheffield Hallam University
Department Materials and Engineering Research Institute (MERI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Investigating to what degree the generation of multiply charged ions by liquid AP-MALDI MS(/MS) will enhance the information content obtainable by MALDI MS with respect to forensic samples.
Collaborator Contribution Providing blood sample standards
Impact So far none - on-going.
Start Year 2017
 
Description Application of liquid AP-MALDI MS to Alzheimer disease diagnosis 
Organisation University of Gothenburg
Department Department of Psychiatry and Neurochemistry
Country Sweden 
Sector Academic/University 
PI Contribution Analysis of the provided samples on our liquid AP-MALDI MS setup
Collaborator Contribution Provided samples
Impact Currently not enough sensitivity for the analysis of compounds of interest at relevant concentrations
Start Year 2016
 
Description Evaluation of liquid AP-MALDI for rapid screening 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution High-throughput AP-MALDI platform was designed. Multiple analytes were analysed in a relevant concentration range. Speed of analysis was assesed.
Collaborator Contribution Analytes were supplied. Data processed.
Impact TBA
Start Year 2016
 
Description Higher order protein structural analysis 
Organisation Laboratory of the Government Chemist (LGC) Ltd
Country United Kingdom 
Sector Private 
PI Contribution Contribution to a multi-lab comparison of higher order protein structural analysis using liquid AP-MALDI MS
Collaborator Contribution Organisation of the multi-lap comparison, including providing and shipping the samples and analysing the data
Impact TBA
Start Year 2015
 
Description Investigation of the oligomerisation extent of KLVFF-COOH and K(Boc)LVFF-NH2 peptides 
Organisation University of Reading
Department Department of Meteorology
Country United Kingdom 
Sector Academic/University 
PI Contribution Tested feasibility of using liquid AP-MALDI MS for the analysis of peptide oligomerisation. Acquired ESI MS data on the peptide oligomerisation. Analysed data and written a contribution to a joint research article.
Collaborator Contribution Designed study. Provided samples. Analyzed data.
Impact Our analysis provided confirmation of a different extent of oligomerisation of two provided samples which was correlated to their biological activity.
Start Year 2016
 
Description Peptidomic analysis of the protein fraction of Stablor® before and after bacterial fermentation 
Organisation LNC Therapeutics
Country France 
Sector Private 
PI Contribution Sample preparation, experimental design and MS analysis and data mining
Collaborator Contribution Provision of raw sample and above funding
Impact MS data. This collaboration is multi-disciplinary, involving scientists from analytical chemistry and food sciences.
Start Year 2018
 
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 Method for Ion Production 
Description Our former IP published under WO2014096847 was assigned to Micromass UK Ltd. 
IP Reference EP13815814 
Protection Patent application published
Year Protection Granted
Licensed Yes
Impact This assignment is part of a wider collaboration between Waters Corporation and our lab.
 
Description Waters Colaborators meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 30 scientists attended the Waters collaborators meeting to share their research and engage in discussion.
Year(s) Of Engagement Activity 2014