Development of a proteomics platform to monitor immune responses in non-mammals.
Lead Research Organisation:
University of Aberdeen
Department Name: College of Life Sci and Med Graduate Sch
Abstract
From early studies of bacterial phagocytosis by sea urchin cells, to the more recent discoveries of B and T cells in chickens or toll-like receptors (TLRs) in fruit flies, the study of immune responses in 'non-traditional' species has greatly enhanced our understanding of the human immune system. While the recent explosion in genomic and transcriptomic sequence data has greatly aided the discovery of orthologues of mammalian immune genes in such species, allowing us to trace their evolutionary histories, our ability to perform functional studies remains limited by to a lack of research reagents (e.g. monoclonal antibodies). In an effort to leap-frog this hurdle we are developing a proteomics platform that will allow researchers to study immune responses in any species where sufficient sequence data is available. The project will involve optimisation of our new platform and assessment of its ability to measure the levels of selected immune proteins in non-mammalian species over the course of an immune response.
Much of our work to date has focused upon the immune system of cartilaginous fishes (sharks, skates, rays and chimera); this group diverged from a common ancestor with other jawed vertebrates around 500 million years ago and are the most ancient species to have a 'mammalian-like' adaptive immune system. As part of an ongoing project we have recently generated a high-coverage, multi-tissue transcriptome for the small-spotted catshark (Scyliorhinus canicula) and are actively mining this dataset for families of immune genes. This places us in a perfect position to assess the 'real-world' utility of our proteomic platform by studying the immune response of immunised catsharks. The project will be extended to additional non-mammalian species either by mining datasets that are already publically-available or following the generation of a transcriptome for a new species.
The student will receive training in standard molecular biology techniques, bioinformatics, in vivo study design and implementation, as well as various immunological assays and techniques. Project-specific proteomics training will be provided through Aberdeen University's Proteomics facility. Training in next-generation sequencing and bioinformatics is available through the recently-established Centre for Genome-Enabled Biology & Medicine (CGEBM). Additional training in generic skills is provided by the CLSM graduate school and the University's Centre for Academic Development.
Achievement of this project will improve our ability to study immune responses in species where functional studies are currently limited by a lack of research reagents. As well as increasing fundamental knowledge regarding the evolution and functioning of the immune system, other potential future applications are diverse and include development of new/improved vaccines for animal health, studying zoonotic infections and host immunity for the protection of human health, and the rapid diagnosis of infectious disease in non-human species.
Much of our work to date has focused upon the immune system of cartilaginous fishes (sharks, skates, rays and chimera); this group diverged from a common ancestor with other jawed vertebrates around 500 million years ago and are the most ancient species to have a 'mammalian-like' adaptive immune system. As part of an ongoing project we have recently generated a high-coverage, multi-tissue transcriptome for the small-spotted catshark (Scyliorhinus canicula) and are actively mining this dataset for families of immune genes. This places us in a perfect position to assess the 'real-world' utility of our proteomic platform by studying the immune response of immunised catsharks. The project will be extended to additional non-mammalian species either by mining datasets that are already publically-available or following the generation of a transcriptome for a new species.
The student will receive training in standard molecular biology techniques, bioinformatics, in vivo study design and implementation, as well as various immunological assays and techniques. Project-specific proteomics training will be provided through Aberdeen University's Proteomics facility. Training in next-generation sequencing and bioinformatics is available through the recently-established Centre for Genome-Enabled Biology & Medicine (CGEBM). Additional training in generic skills is provided by the CLSM graduate school and the University's Centre for Academic Development.
Achievement of this project will improve our ability to study immune responses in species where functional studies are currently limited by a lack of research reagents. As well as increasing fundamental knowledge regarding the evolution and functioning of the immune system, other potential future applications are diverse and include development of new/improved vaccines for animal health, studying zoonotic infections and host immunity for the protection of human health, and the rapid diagnosis of infectious disease in non-human species.
People |
ORCID iD |
| Daniel Macqueen (Primary Supervisor) | |
| David Stead (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M010996/1 | 01/10/2015 | 31/03/2024 | |||
| 1805428 | Studentship | BB/M010996/1 | 01/10/2016 | 31/03/2021 |
| Description | Rainbow trout immune study • Vaccine development to prevent fish diseases in the aquaculture industry typically requires analysis of multiple tissues from large numbers of fish to identify and measure vaccine response. Plasma sampling permits repeat sampling of smaller numbers of live fish to obtain immune protein expression profiles, therefore greatly reducing the need to euthanise large numbers of fish for experimental purposes. • Our study revealed considerable variation in immune response, with individual fish showing very different profiles for many of the plasma proteins analysed. This suggests that much useful information may be being lost with current methodologies which average data from pools of animals. • A subset of proteins called apolipoproteins showed highly coordinated changes in expression across multiple fish and may therefore serve as useful biomarkers of an immune response. • The proteomics dataset generated during this project has been used to understand how the bioinformatics tools used to analyse the detected proteins are able to distinguish duplicated copies of proteins resulting from a salmonid-specific whole-genome duplication event. This will be very useful to other salmonid biologists using LC-MS proteomics in the future. • We are now also using a 'targeted' proteomics approach ('parallel reaction monitoring') to quantify exactly the amounts of selected proteins present at each stage of the immune response. Some of these are present in levels which are too low to be detected using the global approach, but are known to play key roles in the immune response, making them important study targets during vaccine development. Nurse shark immune study • The aim of this part of the project is to characterise and quantify immune protein expression in the nurse shark and then to carry out a comparative study using these data and data obtained from our similar study in rainbow trout. Since minimal genomic data are available for the nurse shark, our first step required assembly and annotation of a de novo nurse shark transcriptome. • Multi-tissue nurse shark samples have been obtained and sequenced using Illumina HiSeq4000 and Pac Bio Sequel, and these have been assembled using the the Trinity hybrid-seq platform. Now that the transcriptome assembly is complete, it is being used to characterise immune protein expression in this species. Baseline plasma proteomic study comparing rainbow trout, nurse shark and sea lamprey (Petromyzon marinus). * A comparative study is being carried out to characterise similarities and differences in immune proteins in the plasma of unstimulated animals from these three species. This builds on the work already carried out using rainbow trout and nurse shark, and enables further investigation into the evolutionary origins of such proteins. |
| Exploitation Route | Mass-spectrometry based proteomics is a developing methodology in the aquaculture industry. We show that it provides a holistic assessment of changes in protein expression during the immune response, including the identification of potential novel biomarkers, making it a valuable technology to those involved in vaccine development for aquaculture. Development of the 'parallel reaction monitoring' method to obtain absolute quantification of immune-relevant proteins will further assist this work. Combined with the use of repeated plasma sampling, this proteomics approach also minimises the need to euthanise large numbers of fish. Our confirmation that that the bioinformatic tools used to analyse the proteins detected are capable of distinguishing duplicated copies of proteins in salmonids will also benefit salmonid biologists using this proteomics approach in the future. |
| Sectors | Agriculture, Food and Drink |
| Description | Travel Award for 14th ISDCI Congress, New Mexico, USA, June 2018. |
| Amount | £420 (GBP) |
| Organisation | Heriot-Watt University |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 06/2018 |
| End | 06/2018 |
| Description | Travel Award for 4th International Conference on Integrative Salmonid Biology, Nov 2019 |
| Amount | £500 (GBP) |
| Organisation | Scottish Aquaculture Innovation Centre |
| Sector | Multiple |
| Country | United Kingdom |
| Start | 11/2019 |
| End | 11/2019 |
| Description | Travel Award for North American Comparative Immunology Workship, Ontario, Canada, June 2019 |
| Amount | $500 (CAD) |
| Organisation | University of Waterloo |
| Sector | Academic/University |
| Country | Canada |
| Start | 06/2019 |
| End | 07/2019 |
| Title | Label free shotgun proteomic method for measuring fish blood plasma proteins after immunization - coupled to repeated sampling of blood from same individual |
| Description | This tool involves the tryptic digestion of a protein sample, before the resultant peptides are injected into and separated by liquid chromatography (we used an UltiMate 3000 RSLCnano system; Dionex/Thermo Scientific) coupled to a Q Exactive Plus quadrupole-equipped orbitrap mass spectrometer (Thermo Scientific). Following mass spectrometry scans, the raw data was analysed using the MaxQuant program (Cox and Mann, 2008; Nat. Biotechnol. 26, 1367-1372), using a label-free quantification method (Cox et al., 2014; Mol. Cell. Proteomics 13, 2513-2526). Identification of proteins was done against the latest rainbow trout reference assembly (GCA_002163495.1). Development of this tool during the award is making a broad positive impact to ongoing research within the groups of the award holders (PI: Macqueen, Former PI: Dooley, Co-I: Stead). The label-free shotgun proteomics approach developed within the project is being used routinely in several projects led by Macqueen to dissect the basis of physiological traits relevant to salmonid aquaculture, and it now a routine method in an armoury of omics tools used by his group. The same approach is being used in a BBSRC PhD studentship (BB/M010996/1) co-supervised with Dooley and Stead. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2016 |
| Provided To Others? | No |
| Impact | A major objective of BB/M026345/1 was to reduce the number of fish used in vaccine testing and immune research. We have successfully developed an approach that combines the use of repeated blood sampling of the same individual during an immune response, with high power proteomics that can measure a large number of proteins simultaneously. This advance allows the generation of more in-depth data regarding the immune response but using far fewer fish. It also gives us direct empirical insights into individual variation in immunological traits missed by more typical experimental designs (i.e. where data is pooled from a large number of animals). Such information will facilitate the identification of variables that separate vaccine-responsive and non-responsive animals, and help inform the design of new and/or more protective, aquaculture vaccines. |
| Title | Technology assay or reagent - Parallel reaction monitoring for measuring fish target blood plasma proteins after immunization - coupled to repeated sampling of blood from same individual |
| Description | A targeted proteomics parallel reaction monitoring (PRM) approach has been developed during the project, with the work led by Co-I Stead, lead Proteomics Technologist within the University of Aberdeen core Proteomics unit. The approach involves the use of heavy-labelled versions of the tryptic peptides targeted by PRM, which are spiked into the sample mix. Fisher Thermo Scientific software PinPoint is used to determine quantitative levels of target proteins. This work is being further developed in a BBSRC PhD studentship, but a publication reporting the findings of the award should be published within 2018. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | Knowledge gained during the award is opening-up a new branch of PRM tools that the Aberdeen core proteomics facility can offer to researchers in the future. A major objective of BB/M026345/1 was to reduce the number of fish used in vaccine testing and immune research. We have successfully developed an approach that combines the use of repeated blood sampling of the same individual during an immune response, with high power proteomics that can measure a large number of proteins simultaneously. This advance allows the generation of more in-depth data regarding the immune response but using far fewer fish. It also gives us direct empirical insights into individual variation in immunological traits missed by more typical experimental designs (i.e. where data is pooled from a large number of animals). Such information will facilitate the identification of variables that separate vaccine-responsive and non-responsive animals, and help inform the design of new and/or more protective, aquaculture vaccines. |
| Title | Nurse shark de novo transcriptome COMPLETE |
| Description | Illumina HiSeq 4000 and PacBio Sequel systems used to sequence multi-tissue nurse shark samples. IDP-denovo sequencing tool used for de novo transcriptome assembly and isoform annotation by hybrid sequencing. This will be used in the future as a reference transcriptome for the proteomics methods used in Research Tools and Methods. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2020 |
| Provided To Others? | No |
| Impact | Will extend the range of currently relatively limited chondrichthyan genomic data and will enable analysis of the chondrichthyan response to an immune challenge. Will provide additional information about the evolution of immune responses. |
| Title | Nurse shark multi-tissue samples for assembly and annotation of a de novo transcriptome (in progress). |
| Description | Illumina HiSeq 4000 and PacBio Sequel systems used to sequence multi-tissue nurse shark samples. IDP-denovo sequencing tool used for de novo transcriptome assembly and isoform annotation by hybrid sequencing. This will be used in the future as a reference transcriptome for the proteomics methods used in Research Tools and Methods. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | Will extend the range of currently relatively limited chondrichthyan genomic data and will enable analysis of the chondrichthyan response to an immune challenge. Will provide additional information about the evolution of immune responses. |
| Description | Collaboration with Helen Dooley lab |
| Organisation | University of Maryland |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | Ongoing shotgun and targeted proteomics work, aiming to understand development of immunity in fishes and sharks using non-lethal methodologies. |
| Collaborator Contribution | Dooley lab has contributed cost of PACBIO (2x SMRT cells) and high coverage Illumina sequencing (2 HiSeq 4000 lanes) of a shark multi-tissue transcriptome, which will form the reference to be used in ongoing proteomics analyses in the Macqueen lab. Dooley lab is also contributing plasma samples gained from a shark long-term immunization study, which will be used for proteomics analysis, in addition to rainbow trout plasma samples. |
| Impact | Talk: Sept 2018. Macqueen DJ. 'Plasma proteomics reveals predominantly individual responses to immunization in rainbow trout'. ARCH-UK annual science event. Sept 3-4 Agri-Food and Biosciences Institute |
| Start Year | 2017 |
| Description | 14th ISDCI Congress, Santa Fe, New Mexico, USA. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster presentation about this project, which increased interest in the use of mass-spectrometry based proteomics approaches and potential future collaborations. |
| Year(s) Of Engagement Activity | 2018 |
| Description | 1st ARCH-UK Annual Science Event, Belfast, UK. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster presentation which increased interest in the use of a mass-spectrometry based approach for vaccine development in aquaculture. |
| Year(s) Of Engagement Activity | 2018 |
| Description | 3rd International Conference on Fish and Shellfish Immunology, Las Palmas de Gran Canaria, Spain. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Oral presentation - abstract accepted. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Conference oral presentation: 'Plasma proteomics reveals predominantly individual responses to immunization in rainbow trout' |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | The purpose was to disseminate applied collaborative proteomics research done in the Macqueen lab at the ARCH-UK annual science event, held on Sept 3-4 at the Agri-Food and Biosciences Institute, Belfast. The title of the talk was "Plasma proteomics reveals predominantly individual responses to immunization in rainbow trout". The meeting was attended by a range of researchers and industrial representatives, focussed on building a sustainable aquacuture indistry in the UK, in addition to a robust research sector contributing to the same aim. My talk was well received, and led to a range of discussions with other researchers about the tools being used in the lab. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://www.aquaculturehub-uk.com/ |
| Description | Coordinated meeting of the international 'Functional Annotation of All Salmonid Genomes' initiative |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | This meeting was attended by 30 salmonid biologists linked to the international FAASG initiative (https://www.faasg.org/). Key discussions were held on the future of the initiative, its links to UK infrastructure (EMBL-EBI) and future funding priorities, influencing funders in attendence (Norwegian Research Council and Genome Canada) |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://icisb.org/faasg-meeting/ |
| Description | Fish Immunology Workshop, Wageningen, Netherlands. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Poster presentation which increased awareness of mass-spectrometry based proteomics as a tool for vaccine development in aquaculture. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Invited talk at International workshop Functional annotation of the Atlantic salmon genome, translation to improved health and performance in aquaculture. 'Advancing aquaculture by genome functional annotation: Memorial University, Canada. Aug 26-27th 2019. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Gave an invited talk at International workshop Functional annotation of the Atlantic salmon genome, translation to improved health and performance in aquaculture. 'Advancing aquaculture by genome functional annotation: Memorial University, Canada. Aug 26-27th 2019. The outcomes were an increased mutual understanding of research and collaborative activity with international collaborating scientists. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Lead coordinator of the fourth International Conference on the Integrative Biology of Salmonids (https://icisb.org/). |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | The fourth International Conference on Integrative Salmonid Biology (ICISB 2019), followed on from previous meetings in 2012 (Oslo, Norway), 2014 (Vancouver, Canada) and 2016 (Puerto Varas, Chile). The ICISB meetings have been core funded and organized by the International Cooperation to Sequence the Atlantic Salmon Genome (ICSASG), a trilateral effort between Canada, Chile and Norway. The theme of ICISB 2019 was'Beyond the genome: taking leaps forward in salmonid biology' to reflect the recent staggering progress in genomic resource development and exploitation since the Atlantic salmon reference genome was published in 2016. There was an audience of ~200, which represented a mixture of researchers from Professors leading in the field, to undergraduate students. Many international collaborations and opportunities for further research, funding and meetings were explored with a range of stakeholders, including funders, media and industry. |
| Year(s) Of Engagement Activity | 2019 |
| URL | https://icisb.org/ |
| Description | Oral presentation at North American Comparative Immunology Workshop, June 2019 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Oral presentation stimulated future collaboration with colleagues carrying out similar work on different species. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Oral presentation delivered online to remotely convened North American Comparative Immunology Workshop (NACIW) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Description of generation of de novo nurse shark transcriptome and study of immunological aspects of the nurse shark plasma proteome. Intended to share knowledge to researchers in the field of comparative immunology. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Poster presentation and participation at 4th International Conference on Integrative Salmonid Biology |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Poster presentation and participation at conference; networking with potential to lead to future employment. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Seminar at the Roslin Institute: "Farmed Fish Integrative Genomics" |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Other audiences |
| Results and Impact | I gave a seminar at the Roslin Institute, which was an overarching overview of the major research projects in my lab. The seminar was linked to job vacancy, so pitched towards the relevance of my work to BBSRC/UKRI remit and the interests/remit of the Roslin Institute. I was succesful in getting the position (Reader, University of Edinburgh), so a major impact followed this seminar. |
| Year(s) Of Engagement Activity | 2018 |