The Next Dimension in Proteomics
Lead Research Organisation:
University of Warwick
Department Name: Chemistry
Abstract
Cells, tissues, and organisms, including humans, are made of a variety of molecules, including proteins. Proteins are the 'molecular machines' responsible for everything a cell does, from replication to processing nutrients. A typical cell might have 10's of thousands of proteins doing myriad particular chemical 'jobs', including synthesizing or recycling other proteins, so the complement of proteins is constantly changing. The field of proteomics involves detecting as many of these proteins as possible, understanding how they change with time and in reaction to stress or chemical treatment. Knowing this information will allow bioscientists, from basic researchers to clinicians, to better understand what is occurring in the cell under conditions of malnutrition, disease, healthy aging, drought, or even pharmaceutical treatment. Developing better methods to measure the 'proteome' accurately and with sufficient detail is a key underpinning research goal which results in better science, better technology, and ultimately better health and healthier food.
We have recently developed a new technology, called 2-dimensional mass spectrometry, which offers the capability to sequence all peptide/protein components in a sample, rather than a select few. We have demonstrated the technology works in several publications and in preliminary data on a whole proteomic analysis of yeast herein, but there are some particular challenges which we must address before the vision can be attained. This proposal aims to solve those challenges.
Firstly, we will optimise our detection and fragmentation methods to increase protein sequence coverage. Secondly, we will fully characterise the technique to determine the sensitivity, resolution, accuracy, dynamic range, and capacity for quantitation and detection of protein modifications. Thirdly, we will develop a set of algorithms which will convert raw 2-dimensional MS data into protein sequence information and integrate those algorithms into a web-based data processing pipeline for automated proteomic data analysis. Finally, we will compare our newly developed 2-dimensional MS proteomic data sets with existing proteomic methodologies on the exact same set of samples, to clearly show the improvements (both delivered and potential) provided by doing proteomics with an extra dimension.
The overall result of this project will be the development of protocols and methods for optimal proteomic analysis using this exciting new technology and a web-based data analysis pipeline which can process the raw 2-dimensional mass spectrometry data into protein sequence information for whole cells and tissues.
We have recently developed a new technology, called 2-dimensional mass spectrometry, which offers the capability to sequence all peptide/protein components in a sample, rather than a select few. We have demonstrated the technology works in several publications and in preliminary data on a whole proteomic analysis of yeast herein, but there are some particular challenges which we must address before the vision can be attained. This proposal aims to solve those challenges.
Firstly, we will optimise our detection and fragmentation methods to increase protein sequence coverage. Secondly, we will fully characterise the technique to determine the sensitivity, resolution, accuracy, dynamic range, and capacity for quantitation and detection of protein modifications. Thirdly, we will develop a set of algorithms which will convert raw 2-dimensional MS data into protein sequence information and integrate those algorithms into a web-based data processing pipeline for automated proteomic data analysis. Finally, we will compare our newly developed 2-dimensional MS proteomic data sets with existing proteomic methodologies on the exact same set of samples, to clearly show the improvements (both delivered and potential) provided by doing proteomics with an extra dimension.
The overall result of this project will be the development of protocols and methods for optimal proteomic analysis using this exciting new technology and a web-based data analysis pipeline which can process the raw 2-dimensional mass spectrometry data into protein sequence information for whole cells and tissues.
Technical Summary
We have recently developed 2-dimensional mass spectrometry (2DMS) to the point where it can now be applied to complex mixtures of proteins and generate unbiased sequence information about all proteins/peptides in proteomics samples. However, for this vision to be realised, a set of challenges must be solved, which is the goal of this proposal.
The test-sample to study for optimizing these methods will be yeast cells, and we will focus on high resolution 2DMS and new fragmentation methods to generate as much information as possible for all the proteins detected in these cells.
Specifically, we will:
1. develop high sequence coverage and depth in proteomic samples using the fragmentation methods of IR-ECD, EID, and UVPD.
2. develop differential extraction protocols which will extract as many detectable proteins and peptides from a sample as possible using solid-phase microextraction and a sample preparation robot.
3. characterise the capabilities of 2DMS with respect to resolution, accuracy, sensitivity, dynamic range, limits of detection, and quantitation.
4. develop top-down methodologies for 2DMS using a combined method called MS/2DMS, and characterise its performance in terms of sequence cleavage coverage and the ability to detect and quantify post-translational modifications.
5. develop algorithms for conversion of raw 2DMS data into protein sequence information including peakpicking, charge-state determination, isotopic distribution alignment, and develop new algorithms for extraction of the new information in 2DMS data contained in neutral-loss lines and precursor scan lines.
6. develop a web-based data analysis pipeline on the Warwick HPC linux clusters for 2DMS data using these algorithms to convert raw data into pseudo MS/MS scan lines for proteomics database search engines.
7. automate the uploading of the peaklists and sequences to online proteomics databases such as PRIDE.
8. directly compare 2DMS with LC-MS/MS for proteomics.
The test-sample to study for optimizing these methods will be yeast cells, and we will focus on high resolution 2DMS and new fragmentation methods to generate as much information as possible for all the proteins detected in these cells.
Specifically, we will:
1. develop high sequence coverage and depth in proteomic samples using the fragmentation methods of IR-ECD, EID, and UVPD.
2. develop differential extraction protocols which will extract as many detectable proteins and peptides from a sample as possible using solid-phase microextraction and a sample preparation robot.
3. characterise the capabilities of 2DMS with respect to resolution, accuracy, sensitivity, dynamic range, limits of detection, and quantitation.
4. develop top-down methodologies for 2DMS using a combined method called MS/2DMS, and characterise its performance in terms of sequence cleavage coverage and the ability to detect and quantify post-translational modifications.
5. develop algorithms for conversion of raw 2DMS data into protein sequence information including peakpicking, charge-state determination, isotopic distribution alignment, and develop new algorithms for extraction of the new information in 2DMS data contained in neutral-loss lines and precursor scan lines.
6. develop a web-based data analysis pipeline on the Warwick HPC linux clusters for 2DMS data using these algorithms to convert raw data into pseudo MS/MS scan lines for proteomics database search engines.
7. automate the uploading of the peaklists and sequences to online proteomics databases such as PRIDE.
8. directly compare 2DMS with LC-MS/MS for proteomics.
Planned Impact
We have recently developed an exciting new 2-dimensional mass spectrometry (2DMS) technology that can be applied in the area of proteomics, provided the challenges discussed herein are solved.
Economy
The proteomics market is substantial, with the annual market for proteomics equipment alone was >$10 billion in 2013 and is expected to reach >$20 billion by 2018. The total research and development effort using proteomics is far larger as these are fundamental tools that allow industrial and research scientists to detect proteins and protein changes in cells as a response to stimuli such as pharmaceutical treatment, disease, or drought. Furthermore, the recent results from various proteomics conferences (including ASMS, HUPO, and the recent BSPR conference in Reading) show that a series of new and credible biomarkers are being developed based on modern proteomics techniques. Fundamental new technologies, like 2DMS, will have a large, long-term impact.
New developments in 2DMS technology, such as those discussed herein, are likely to generate new intellectual property, which can be patented and licensed to commercial agencies such as Bruker UK, Ltd. As it currently exists, the 2DMS technique can already be implemented on any FTICR mass spectrometer, but the bottleneck is in the data processing. The data processing software will clearly also have intellectual property associated with it which can be licensed for further commercial impact.
People:
Economically, this project will train two PDRAs specifically, and those PDRAs will help to train PhD, MSc, and undergraduate project students in the methods of advanced mass spectrometry and proteomics. These trained personnel are a currently limiting resource for the UK bio-pharma, clinical, and medical industries, and they will have a large, long-term impact on that industry. The PDRA's will also be trained in science engagement with the public as part of their outreach activities described in the 'Pathways to Impact' plan.
Science:
Overall, the 2DMS developments proposed herein will improve proteomics, which will improve data quality and analysis for proteomic samples throughout the biochemistry, pharmaceutical, medical, and clinical fields. The ability to know precisely which proteins are present and how they are modified will have a large impact scientifically.
Society:
More specifically, as deliverables for the 'Pathways to Impact Plan', this project will provide a series of software packages and tools for researchers in this area, will provide new teaching materials in mass spectrometry related to 2-dimensional MS techniques, and will generate an internet video of the experiment and technique to help train the next generation of students. Additionally, we will collaborate with our industrial partners to support CASE and other industrially funded PhD students to train those students on samples of interest to our partners, and thereby get those same partners interested and integrated into using these advanced mass spectrometry techniques.
Finally, the ultimate benefit of this research is that it will generate better instruments and methods to generate better proteomic data. This will lead on to better understanding of the underlying causes of aging, health, and disease. That understanding will lead to improved healthcare, diet, and disease treatment which will result in longer, healthier lives.
Economy
The proteomics market is substantial, with the annual market for proteomics equipment alone was >$10 billion in 2013 and is expected to reach >$20 billion by 2018. The total research and development effort using proteomics is far larger as these are fundamental tools that allow industrial and research scientists to detect proteins and protein changes in cells as a response to stimuli such as pharmaceutical treatment, disease, or drought. Furthermore, the recent results from various proteomics conferences (including ASMS, HUPO, and the recent BSPR conference in Reading) show that a series of new and credible biomarkers are being developed based on modern proteomics techniques. Fundamental new technologies, like 2DMS, will have a large, long-term impact.
New developments in 2DMS technology, such as those discussed herein, are likely to generate new intellectual property, which can be patented and licensed to commercial agencies such as Bruker UK, Ltd. As it currently exists, the 2DMS technique can already be implemented on any FTICR mass spectrometer, but the bottleneck is in the data processing. The data processing software will clearly also have intellectual property associated with it which can be licensed for further commercial impact.
People:
Economically, this project will train two PDRAs specifically, and those PDRAs will help to train PhD, MSc, and undergraduate project students in the methods of advanced mass spectrometry and proteomics. These trained personnel are a currently limiting resource for the UK bio-pharma, clinical, and medical industries, and they will have a large, long-term impact on that industry. The PDRA's will also be trained in science engagement with the public as part of their outreach activities described in the 'Pathways to Impact' plan.
Science:
Overall, the 2DMS developments proposed herein will improve proteomics, which will improve data quality and analysis for proteomic samples throughout the biochemistry, pharmaceutical, medical, and clinical fields. The ability to know precisely which proteins are present and how they are modified will have a large impact scientifically.
Society:
More specifically, as deliverables for the 'Pathways to Impact Plan', this project will provide a series of software packages and tools for researchers in this area, will provide new teaching materials in mass spectrometry related to 2-dimensional MS techniques, and will generate an internet video of the experiment and technique to help train the next generation of students. Additionally, we will collaborate with our industrial partners to support CASE and other industrially funded PhD students to train those students on samples of interest to our partners, and thereby get those same partners interested and integrated into using these advanced mass spectrometry techniques.
Finally, the ultimate benefit of this research is that it will generate better instruments and methods to generate better proteomic data. This will lead on to better understanding of the underlying causes of aging, health, and disease. That understanding will lead to improved healthcare, diet, and disease treatment which will result in longer, healthier lives.
Organisations
People |
ORCID iD |
Peter O'Connor (Principal Investigator) |
Publications
Aliakbari F
(2020)
Multiple Protective Roles of Nanoliposome-Incorporated Baicalein against Alpha-Synuclein Aggregates
in Advanced Functional Materials
Alostad LK
(2022)
Investigating the Influence of n-Heptane versus n-Nonane upon the Extraction of Asphaltenes.
in Energy & fuels : an American Chemical Society journal
Banerjee S
(2018)
New activation mechanism for half-sandwich organometallic anticancer complexes.
in Chemical science
Banerjee S
(2019)
Generation of maghemite nanocrystals from iron-sulfur centres.
in Dalton transactions (Cambridge, England : 2003)
Brooks J
(2020)
Label-Free Nanoimaging of Neuromelanin in the Brain by Soft X-ray Spectromicroscopy.
in Angewandte Chemie (International ed. in English)
Brooks J
(2020)
Label-Free Nanoimaging of Neuromelanin in the Brain by Soft X-ray Spectromicroscopy
in Angewandte Chemie
Chen P
(2019)
Discovery of novel, potent, isosteviol-based antithrombotic agents.
in European journal of medicinal chemistry
Chiu C
(2020)
Metallocomplex-Peptide Interactions Studied by Ultrahigh Resolution Mass Spectrometry
in Journal of the American Society for Mass Spectrometry
Donnelly JM
(2021)
Cu(III)-bis-thiolato complex forms an unusual mono-thiolato Cu(III)-peroxido adduct.
in Chemical communications (Cambridge, England)
Floris F
(2018)
Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.
in Journal of the American Society for Mass Spectrometry
Floris F
(2018)
Top-Down Deep Sequencing of Ubiquitin Using Two-Dimensional Mass Spectrometry
in Analytical Chemistry
Floris F
(2017)
Bottom-Up Two-Dimensional Electron-Capture Dissociation Mass Spectrometry of Calmodulin
in Journal of the American Society for Mass Spectrometry
Floris F
(2017)
Polymer Analysis in the Second Dimension: Preliminary Studies for the Characterization of Polymers with 2D MS.
in Analytical chemistry
Haris A
(2022)
Differentiation of Dihydroxylated Vitamin D3 Isomers Using Tandem Mass Spectrometry.
in Journal of the American Society for Mass Spectrometry
Imberti C
(2021)
Facile protein conjugation of platinum for light-activated cytotoxic payload release.
in Chemical communications (Cambridge, England)
Lam Y
(2020)
Does deamidation affect inhibitory mechanisms towards amyloid protein aggregation?
in Chemical Communications
Lam YPY
(2020)
Determination of the Aggregate Binding Site of Amyloid Protofibrils Using Electron Capture Dissociation Tandem Mass Spectrometry.
in Journal of the American Society for Mass Spectrometry
Lam YPY
(2018)
Does deamidation of islet amyloid polypeptide accelerate amyloid fibril formation?
in Chemical communications (Cambridge, England)
Lermyte F
(2019)
Metal Ion Binding to the Amyloid ß Monomer Studied by Native Top-Down FTICR Mass Spectrometry.
in Journal of the American Society for Mass Spectrometry
Lermyte F
(2019)
MIND: A Double-Linear Model To Accurately Determine Monoisotopic Precursor Mass in High-Resolution Top-Down Proteomics.
in Analytical chemistry
Lermyte F
(2019)
Top or Middle? Up or Down? Toward a Standard Lexicon for Protein Top-Down and Allied Mass Spectrometry Approaches.
in Journal of the American Society for Mass Spectrometry
Lermyte F
(2021)
Solution Condition-Dependent Formation of Gas-Phase Protomers of Alpha-Synuclein in Electrospray Ionization.
in Journal of the American Society for Mass Spectrometry
Lermyte F
(2019)
Emerging Approaches to Investigate the Influence of Transition Metals in the Proteinopathies.
in Cells
Littlejohn C
(2023)
In Silico Demonstration of Two-Dimensional Mass Spectrometry Using Spatially Dependent Fragmentation.
in Journal of the American Society for Mass Spectrometry
Marzullo BP
(2020)
Advantages of Two-Dimensional Electron-Induced Dissociation and Infrared Multiphoton Dissociation Mass Spectrometry for the Analysis of Agrochemicals.
in Analytical chemistry
Marzullo BP
(2021)
Combining Ultraviolet Photodissociation and Two-Dimensional Mass Spectrometry: A Contemporary Approach for Characterizing Singly Charged Agrochemicals.
in Analytical chemistry
Marzullo BP
(2020)
Comparison of Fragmentation Techniques for the Structural Characterization of Singly Charged Agrochemicals.
in Analytical chemistry
Morgan TE
(2018)
Coupling Electron Capture Dissociation and the Modified Kendrick Mass Defect for Sequencing of a Poly(2-ethyl-2-oxazoline) Polymer.
in Analytical chemistry
Morgan TE
(2020)
Electron Capture Dissociation of Trithiocarbonate-Terminated Acrylamide Homo- and Copolymers: A Terminus-Directed Mechanism?
in Analytical chemistry
Morgan TE
(2022)
Stochasticity of poly(2-oxazoline) oligomer hydrolysis determined by tandem mass spectrometry.
in Polymer chemistry
Palacio Lozano DC
(2022)
Revealing the Reactivity of Individual Chemical Entities in Complex Mixtures: the Chemistry Behind Bio-Oil Upgrading.
in Analytical chemistry
Paris J
(2022)
Multimodal Tandem Mass Spectrometry Techniques for the Analysis of Phosphopeptides.
in Journal of the American Society for Mass Spectrometry
Paris J
(2020)
Facile Determination of Phosphorylation Sites in Peptides Using Two-Dimensional Mass Spectrometry.
in Analytical chemistry
Paris J
(2021)
Two-Dimensional Mass Spectrometry Analysis of IgG1 Antibodies.
in Journal of the American Society for Mass Spectrometry
Rüger C
(2017)
Comprehensive chemical comparison of fuel composition and aerosol particles emitted from a ship diesel engine by gas chromatography atmospheric pressure chemical ionisation ultra-high resolution mass spectrometry with improved data processing routines
in European Journal of Mass Spectrometry
Theisen A
(2022)
Enhancing Biomolecule Analysis and 2DMS Experiments by Implementation of (Activated Ion) 193 nm UVPD on a FT-ICR Mass Spectrometer.
in Analytical chemistry
Van Agthoven MA
(2019)
Phase relationships in two-dimensional mass spectrometry.
in Journal of the American Society for Mass Spectrometry
Van Agthoven MA
(2017)
Two-dimensional mass spectrometry in a linear ion trap, an in silico model.
in Rapid communications in mass spectrometry : RCM
Van Agthoven MA
(2019)
Two-dimensional mass spectrometry: new perspectives for tandem mass spectrometry.
in European biophysics journal : EBJ
Van Agthoven MA
(2018)
Can Two-Dimensional IR-ECD Mass Spectrometry Improve Peptide de Novo Sequencing?
in Analytical chemistry
Wootton C
(2019)
Structural analysis of peptides modified with organo-iridium complexes, opportunities from multi-mode fragmentation
in The Analyst
Wootton CA
(2017)
Automatic assignment of metal-containing peptides in proteomic LC-MS and MS/MS data sets.
in The Analyst
Wootton CA
(2018)
Sequence-dependent attack on peptides by photoactivated platinum anticancer complexes.
in Chemical science
Zhang P
(2017)
Innentitelbild: Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells (Angew. Chem. 47/2017)
in Angewandte Chemie
Zhang P
(2017)
Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells.
in Angewandte Chemie (International ed. in English)
Zhang P
(2017)
Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells
in Angewandte Chemie
Zhang P
(2017)
Inside Cover: Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells (Angew. Chem. Int. Ed. 47/2017)
in Angewandte Chemie International Edition
Description | This is an ongoing grant, so key findings are preliminary, but we have shown that two dimensional mass spectrometry is applicable to proteomics. |
Exploitation Route | It's too early to say, but it's led to a spinout company, as listed. |
Sectors | Agriculture, Food and Drink,Chemicals,Digital/Communication/Information Technologies (including Software),Education,Electronics,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | We have started a spinout company based on these results. As listed. |
First Year Of Impact | 2017 |
Sector | Chemicals,Digital/Communication/Information Technologies (including Software),Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Title | ???? |
Description | A method of carrying out mass spectrometry comprises: using an electrostatic or electrodynamic ion trap to contain a plurality of ions, each ion having a mass to charge ratio, the ions having a firstplurality of mass to charge ratios, each ion following a path within the electrostatic or electrodynamic ion trap having a radius; and for each of a second plurality of the mass to charge ratios: modulating the radii of the ions in a mass to charge ratio-dependent fashion dependent upon the mass to charge ratio; fragmenting the ions thus modulated in a radius-dependent fashion; and determining a mass spectrum of the ions. |
IP Reference | CN109937465 |
Protection | Patent granted |
Year Protection Granted | 2019 |
Licensed | Yes |
Impact | This patent led to the creation of a spinout company, Verdel Instruments Ltd. |
Title | MASS SPECTROMETRY |
Description | A method of carrying out mass spectrometry, comprising: using an electrostatic or electrodynamic ion trap to contain a plurality of ions, each ion having a mass to charge ratio, the ions having a first plurality of mass to charge ratios, each ion following a path within the electrostatic or electrodynamic ion trap having a radius; and for each of a second plurality of the mass to charge ratios: modulating the radii of the ions in a mass to charge ratio-dependent fashion dependent upon the mass to charge ratio; fragmenting the ions thus modulated in a radius-dependent fashion; and determining a mass spectrum of the ions. |
IP Reference | WO2018046968 |
Protection | Patent granted |
Year Protection Granted | 2018 |
Licensed | Yes |
Impact | This patent led to the creation of Verdel Instruments Ltd, as a spinout company. |
Title | MASS SPECTROMETRY |
Description | We developed 2DMS in a linear ion trap. |
IP Reference | EP3510629 |
Protection | Patent granted |
Year Protection Granted | 2019 |
Licensed | Yes |
Impact | This patent led to the creation of Verdel Instruments Ltd, as a spinout company. |
Company Name | VERDEL INSTRUMENTS LTD |
Description | This is a spinout company based on a patent which we filed from the BBSRC and EPSRC funded research mentioned. The company aims to develop a new type of mass spectrometer. |
Year Established | 2018 |
Impact | We've obtained the match funding necessary. |