Keeping up with the bases: 'nextgen' approaches to proteomics
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
A famous biochemist, Arthur Kornberg, in his book "For the Love of Enzymes" once said 'DNA and RNA are the script, but proteins are the actors'. Proteomics, the study of the actors, (whether in soliloquy or in crowd scenes) has terrific potential in diagnostics, in the analysis of new disease biomarkers, in understanding the fundamental ways by which the 'intent' of the genes is realised. At present, the global study of proteins (proteomics) is lagging behind our understanding of genomes and RNA, and without radical new technical approaches, taking the best of the analytical capability and coupling it to new methods of sample delivery, the gap is likely to widen.
The challenges are several. First, a true global proteome analysis has to be able to deal with a highly complex mixture of proteins, some present in huge quantities, others at vanishingly low levels. This will require a degree of normalization, in which low abundance proteins are brought to the analytical step in sufficient amounts for analysis, and high abundant proteins are non-selectively sampled.
Secondly, current proteomics is still predominantly based on prior digestion of proteins to multiple smaller peptides using an enzyme (trypsin) derived from the gut. This not only increases analyte complexity about 50 times but also conceals much of the subtlety of the protein world (just as a pile of bricks cannot inform about the structure of the the many building types and variants that could have been made from those bricks). A future solution should be based on protein-level analysis - architecture is less about the study of bricks than it is the exploration and celebration of the entire structures that the bricks are assembled to create.
Finally, we deliver peptides slowly (1-4h per sample) by rather troublesome chromatography. Alternative approaches to protein-level delivery are required.
I propose that we should plan to analyse a proteome without the complication of digestion or of chromatography. This poses new challenges, because we cannot expect the mass spectrometer to be able to analyse a whole proteome at once (it is just too complex). I therefore wish to devise an entirely new approach to proteome analysis based on delivery of a small number of proteins at any one time to the analytical platforms.
The industrial collaborator has invented new types of genetically altered proteins ('Affimers', because they have a high affinity for selected target proteins) that are capable of selectively binding and fishing out a few proteins at a time. With appropriate analytical instrumentation, we should then be able to deliver the payload (proteins) in such a way that we can analyse them directly, capturing all of the complexity of the protein world - the architect's view. In the longer term, engineering solutions to payload delivery could make this the preferred approach to proteome analysis.
The challenges are several. First, a true global proteome analysis has to be able to deal with a highly complex mixture of proteins, some present in huge quantities, others at vanishingly low levels. This will require a degree of normalization, in which low abundance proteins are brought to the analytical step in sufficient amounts for analysis, and high abundant proteins are non-selectively sampled.
Secondly, current proteomics is still predominantly based on prior digestion of proteins to multiple smaller peptides using an enzyme (trypsin) derived from the gut. This not only increases analyte complexity about 50 times but also conceals much of the subtlety of the protein world (just as a pile of bricks cannot inform about the structure of the the many building types and variants that could have been made from those bricks). A future solution should be based on protein-level analysis - architecture is less about the study of bricks than it is the exploration and celebration of the entire structures that the bricks are assembled to create.
Finally, we deliver peptides slowly (1-4h per sample) by rather troublesome chromatography. Alternative approaches to protein-level delivery are required.
I propose that we should plan to analyse a proteome without the complication of digestion or of chromatography. This poses new challenges, because we cannot expect the mass spectrometer to be able to analyse a whole proteome at once (it is just too complex). I therefore wish to devise an entirely new approach to proteome analysis based on delivery of a small number of proteins at any one time to the analytical platforms.
The industrial collaborator has invented new types of genetically altered proteins ('Affimers', because they have a high affinity for selected target proteins) that are capable of selectively binding and fishing out a few proteins at a time. With appropriate analytical instrumentation, we should then be able to deliver the payload (proteins) in such a way that we can analyse them directly, capturing all of the complexity of the protein world - the architect's view. In the longer term, engineering solutions to payload delivery could make this the preferred approach to proteome analysis.
Technical Summary
I will explore the potential of combinatorial protein binding affinity reagents for proteome simplification and top-down mass spectrometric characterisation. Properly developed, this will enhance our ability to explore proteomes, and to capture the complexity of protein modifications and decorations at the protein level.
Avacta has new 'Affimer' technology (libraries of specific and stable protein binders) that will be brought to bear on this problem. I will explore the broad applicability of Affimers as highly selective and clean reagents for subsampling of a proteome, using bacterial, eukaryotic and secretome samples as 'proof of principle' analytes. The first two will be labelled by stable isotopes to provide an independent confirmation of the selectivity and specificity of the Affimers, provide by Avacta, one of the industrial collaborators.
Affimer arrays containing 16,0000 or 100,000 different Affimers will be exposed to differential proteomes, labelled with fluorescent tags in vitro. The array positions showing differential fluorescence will be used to identify the corresponding Affimer, that will then be prepared in nanomol (10-100 microgram) quantities to allow capture of protein payloads in quantities commensurate with discovery by bottom-up proteomics, in addition to defining the extent and variability in background. When the identifies of the protein payloads have been acquired, the same Affimers will be used in a top-down proteomics workflow, using high resolution instrumentation and a new electron transfer dissociation source at my other Industrial Partner (Waters).
I will understand the extent to which Affimers can be used as selective proteome capture reagents, and in particular whether a combination of capture and top-down proteomics can uncover the complexity of the proteome in protein space that is lost when bottom-up workflows are employed, as proteases decouple the connectivity between for example, post-translational modifications.
Avacta has new 'Affimer' technology (libraries of specific and stable protein binders) that will be brought to bear on this problem. I will explore the broad applicability of Affimers as highly selective and clean reagents for subsampling of a proteome, using bacterial, eukaryotic and secretome samples as 'proof of principle' analytes. The first two will be labelled by stable isotopes to provide an independent confirmation of the selectivity and specificity of the Affimers, provide by Avacta, one of the industrial collaborators.
Affimer arrays containing 16,0000 or 100,000 different Affimers will be exposed to differential proteomes, labelled with fluorescent tags in vitro. The array positions showing differential fluorescence will be used to identify the corresponding Affimer, that will then be prepared in nanomol (10-100 microgram) quantities to allow capture of protein payloads in quantities commensurate with discovery by bottom-up proteomics, in addition to defining the extent and variability in background. When the identifies of the protein payloads have been acquired, the same Affimers will be used in a top-down proteomics workflow, using high resolution instrumentation and a new electron transfer dissociation source at my other Industrial Partner (Waters).
I will understand the extent to which Affimers can be used as selective proteome capture reagents, and in particular whether a combination of capture and top-down proteomics can uncover the complexity of the proteome in protein space that is lost when bottom-up workflows are employed, as proteases decouple the connectivity between for example, post-translational modifications.
Planned Impact
Industrial engagement
This programme of research will bring together an academic research group (led by the applicant), a company specialising in protein binders (Avacta) and a mass spectrometry company with expertise in sample delivery and analysis (Waters). The engagement of Waters is assured through the applicant's Industrial Fellowship, but the engagement of Avacta, the specific purpose of this proposal will ensure that all components are in place for a highly productive collaboration.
The work could lead to new panels of binders and new technologies for high throughput delivery of payloads to the mass spectrometer, both of which have terrific potential for commercialisation.
I anticipate that new products and technologies will be a direct result of this research. The programme here is pre-translational and will establish important proofs of concept that will seed the next phase of development. The commitment of Avacta is specified in the proposal, the engagement of Waters is implicit through my Fellowship. Thus, I expect significant commercial development to ensue from this work
Economic and Societal Impacts
If successful, this work will generate new modalities for the analysis of biomarker panels for rapid screening of health, or for environmental screening, as exemplars. The ability to 'go fishing' for specific reporter molecules and then analyse them, in all their subtlety, is something that has not yet been achieved, and could have considerable economic significance.
Academic Impacts
The main academic impact of this work lies in the exploration of new approaches to high throughput, reliable proteomics. This work could lead to new proteomics-mediated discoveries across the whole of the biological and biomedical arena (see the section on Academic Beneficiaries).
This programme of research will bring together an academic research group (led by the applicant), a company specialising in protein binders (Avacta) and a mass spectrometry company with expertise in sample delivery and analysis (Waters). The engagement of Waters is assured through the applicant's Industrial Fellowship, but the engagement of Avacta, the specific purpose of this proposal will ensure that all components are in place for a highly productive collaboration.
The work could lead to new panels of binders and new technologies for high throughput delivery of payloads to the mass spectrometer, both of which have terrific potential for commercialisation.
I anticipate that new products and technologies will be a direct result of this research. The programme here is pre-translational and will establish important proofs of concept that will seed the next phase of development. The commitment of Avacta is specified in the proposal, the engagement of Waters is implicit through my Fellowship. Thus, I expect significant commercial development to ensue from this work
Economic and Societal Impacts
If successful, this work will generate new modalities for the analysis of biomarker panels for rapid screening of health, or for environmental screening, as exemplars. The ability to 'go fishing' for specific reporter molecules and then analyse them, in all their subtlety, is something that has not yet been achieved, and could have considerable economic significance.
Academic Impacts
The main academic impact of this work lies in the exploration of new approaches to high throughput, reliable proteomics. This work could lead to new proteomics-mediated discoveries across the whole of the biological and biomedical arena (see the section on Academic Beneficiaries).
People |
ORCID iD |
| Robert Beynon (Principal Investigator) |
| Description | Exploration of routes to rational simplification of complex protein mixtures, potentially leading to new ways of measuring complex mixtures with higher speed and greater confidence However, through this work is has become clear that the large scale construction of panels of affimers for proteome resolution and simplification is a major challenge, and is unlikely to be a solution to accessing the 'dark proteome'. A key factor in development of the fellowship subject is the development of combinatorial libraries for selective abstraction and delivery of rationally reduced proteomes to a mass spectrometer. In the application, I proposed using two approaches, based either on hexapeptide libraries or on scannins, combinatorially variable proteins with hypervariable protein binding loops assembled on a rigid scaffold. The scannins were developed by Dr Paul Ko Ferrigno at Leeds University, but by the time the fellowship had started, Dr Ko Ferrigno's start up company, Aptuscan, had been acquired by a larger company, Avacta, and Dr Ko Ferrigno has left The Universty to lead the scannin development (renamed 'Affimers') at Aptuscan. Although Dr Ko Ferrigno was keen to continue our association, the realtinoship between the main industrial partner, Waters, Aptuscan and the University of Liverpool need clarification, and it took a little time before the relevant paperwork was in place, protecting IP and permitting free discussion between the three partners. Avacta and I were able to submit an application for a BBSRC Industrial CASE studentship, a competitive award process. |
| Exploitation Route | Once published (after IP exploration), we thought this research could be widely adopted. We used two panels of second generation affimers to understand heir scope and applicability in proteome desegregation. We now know that there is no easy route to affimer generation at this scale, and that the possibility of IP developmet is very slim. |
| Sectors | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Not yet broadly taken up. Very much pre-deployment research. However, we are seeking to gain additional funding with second generation affimers to approach the strategy of selective dissection of the proteome. |
| First Year Of Impact | 2014 |
| Title | New affiity reagent for biomarker detection |
| Description | New adhirons to biomarkers of reflux. |
| Type Of Material | Technology assay or reagent |
| Provided To Others? | No |
| Impact | If adhirons are effective, we will have a radically improved test for a 'difficult' biomarker. |