Mapping protein 'interactomes' within membrane trafficking pathways: combining mass spectrometry and SILAC labelling with targeted tyramine tagging
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
Eukaryotic cells (ie those cells with nuclei) contain a rich collection of internal membrane-bound compartments. These compartments often contain specialized proteins and serve many purposes vital for the well-being of the cell. For example, we are beginning to appreciate that protein mis-targeting within these pathways underlie a number of important diseases. A major area of active research within modern cell biology is to understand how these targeting events take place. In general terms, proteins are selectively targeted to different locations because they interact with 'coat proteins' that capture and direct their targets to different internal compartments. During this process, the coat proteins transiently assemble onto the internal membranes. Furthermore, many of the proteins that are selectively targeted also occur in discrete 'patches' within the targeted membrane themselves, and so the local molecular neighbours may also affect these interactions.
Unfortunately, there is still much uncertainty concerning the nature of some of these protein complexes. Among the many technical problems we face when trying to characterize them is the fact that many interact with their targets only fleetingly and with relatively low concentrations and/or low affinity for targets. It should be noted that this is a general problem that occurs in other aspects of cell biology. For example, hormones such as insulin trigger the assembly of specific proteins onto internal membrane compartments and again these have been difficult to characterize for similar reasons.
Here we propose a method to address this general problem. It develops and extends techniques that have been successfully used in other contexts, but not yet in this combination. Briefly, we will use a suitable protein labeled with an enzyme called peroxidase. This enzyme can convert a chemical called tyramine into a very unstable reagent that will only 'tag' molecules in the immediate vicinity of the enzyme. A suitable protein, labeled with peroxidase will be introduced into cells. Then tyramine reagent will be added to 'tag' both the protein and its immediate neighbours. These molecules can then be recognized and purified by their specific 'tyramine tag'. Once purified, they will be identified by a method called mass spectrometry that can successfully characterize large numbers of proteins in complex mixtures. In addition, we propose to include specifically adapted software that will enable the easy and accurate analysis of any data obtained with the technique. We believe that our process, that we call 'targeted tyramine tagging' will offer a significant improvement in the ability to identify transiently-interacting protein-protein partners within the cell.
Unfortunately, there is still much uncertainty concerning the nature of some of these protein complexes. Among the many technical problems we face when trying to characterize them is the fact that many interact with their targets only fleetingly and with relatively low concentrations and/or low affinity for targets. It should be noted that this is a general problem that occurs in other aspects of cell biology. For example, hormones such as insulin trigger the assembly of specific proteins onto internal membrane compartments and again these have been difficult to characterize for similar reasons.
Here we propose a method to address this general problem. It develops and extends techniques that have been successfully used in other contexts, but not yet in this combination. Briefly, we will use a suitable protein labeled with an enzyme called peroxidase. This enzyme can convert a chemical called tyramine into a very unstable reagent that will only 'tag' molecules in the immediate vicinity of the enzyme. A suitable protein, labeled with peroxidase will be introduced into cells. Then tyramine reagent will be added to 'tag' both the protein and its immediate neighbours. These molecules can then be recognized and purified by their specific 'tyramine tag'. Once purified, they will be identified by a method called mass spectrometry that can successfully characterize large numbers of proteins in complex mixtures. In addition, we propose to include specifically adapted software that will enable the easy and accurate analysis of any data obtained with the technique. We believe that our process, that we call 'targeted tyramine tagging' will offer a significant improvement in the ability to identify transiently-interacting protein-protein partners within the cell.
Technical Summary
Emergent behavior in cell biology often relies on the selective interactions of protein assemblies on internal membrane surfaces. Examples include the formation of coat proteins onto endosomes, and the assembly of protein complexes in response to signal transduction events. These interactions are difficult to examine using current proteomic methods because they are transient, dynamic and occur with low affinity on two dimensional membrane surfaces that are not stable following cell lysis.
A general proteomic method to address this problem would be of considerable interest to the biochemical, cell biology and proteomics communities. Here we propose the combined use of 'target tyramine tagging' together with high throughput SILAC mass spectrometry. This method is based on our recent experience to selectively label protein assemblies at the cell surface. We believe it can now be generalized to examine other protein 'interactomes' throughout the cell.
As 'proof of principle' we will examine the recycling pathway taken by transferrin through endosomes. We will add peroxidase-labeled transferrin to cells so as to fill internal compartments. Then we will add a membrane-permeant tyramine-biotin or tyramine-fluorescein derivative. When acted on by peroxidase, tyramine covalently labels tyrosine residues in proteins immediately adjacent to the peroxidase. Hence, the 'tyramine tagged' proteins can be isolated by standard affinity capture and characterized by mass spectrometry. We will combine this 'targeted tyramine tagging' with SILAC labeling to discriminate specific and non-specifically tagged proteins. We will explore the potential of this method when combined with SILAC labeling, to provide kinetic data on different proteins encountered by transferrin on its sequential journey through endosome compartments.
An integral part of our proposal is also to adapt associated software for the facile analysis of data generated by this method.
A general proteomic method to address this problem would be of considerable interest to the biochemical, cell biology and proteomics communities. Here we propose the combined use of 'target tyramine tagging' together with high throughput SILAC mass spectrometry. This method is based on our recent experience to selectively label protein assemblies at the cell surface. We believe it can now be generalized to examine other protein 'interactomes' throughout the cell.
As 'proof of principle' we will examine the recycling pathway taken by transferrin through endosomes. We will add peroxidase-labeled transferrin to cells so as to fill internal compartments. Then we will add a membrane-permeant tyramine-biotin or tyramine-fluorescein derivative. When acted on by peroxidase, tyramine covalently labels tyrosine residues in proteins immediately adjacent to the peroxidase. Hence, the 'tyramine tagged' proteins can be isolated by standard affinity capture and characterized by mass spectrometry. We will combine this 'targeted tyramine tagging' with SILAC labeling to discriminate specific and non-specifically tagged proteins. We will explore the potential of this method when combined with SILAC labeling, to provide kinetic data on different proteins encountered by transferrin on its sequential journey through endosome compartments.
An integral part of our proposal is also to adapt associated software for the facile analysis of data generated by this method.
Planned Impact
The man impacts expected form this project will be:
(i)Biochemistry/cell biology community: The development of the 'targeted tyramine tagging' methodology in conjunction with SILAC mass spectrometry will facilitate the analysis of intramembrane protein clusters. Such localized interactions lie at the explanatory heart of many key biochemical and cell biological processes including, but not limited to: membrane trafficking, cell metabolism and signal transduction. Developing accurate, sensitive and robust proteomic methods, including associated software for the analysis and characterisation of these complexes is a major priority.
(ii)Proteomics community (both academic and industrial): There are already close collaborations between the University of Cambridge and several mass spectrometry vendors and proteomics software vendors including Thermo Finnigan, Applied Biosystems, Waters and Matrix Science. The proteomics community as a whole will benefit from the development of such widely applicable methodology and associated software. KSL has collaborated with Matrix Science and Applied Biosystems for many years including Applied Biosystems contribution to a BBSRC award which resulted in the first developments of organelle proteomics methods. We envisage that such collaborations with mass spectrometry vendors will continue throughout the period of this grant, and will result in us working with the appropriate vendors to incorporate them into the workflow.
(iii)Pharmaceutical industry: The APJ and KSL have given talks frequently to the pharmaceutical industry on cell biology, ion channels and proteomics, including Glaxo Smith Kline and Pfizer. We have numerous contacts with these industrial research groups, including the newly established Pfizer research groups at Granta Park Cambridge. KSL has discussed proteomic methodology with Glaxo Smith Kline, Astra Zeneca and also Genentech. The methodologies developed within the context of this proposal are so fundamental that they could underpin many area of pharmaceutical research.
(iv)Full descriptions of this work will be published in peer reviewed literature and presented at academic conferences.
(v) Software developed as part of this project will be freely disseminated to the scientific community.
(i)Biochemistry/cell biology community: The development of the 'targeted tyramine tagging' methodology in conjunction with SILAC mass spectrometry will facilitate the analysis of intramembrane protein clusters. Such localized interactions lie at the explanatory heart of many key biochemical and cell biological processes including, but not limited to: membrane trafficking, cell metabolism and signal transduction. Developing accurate, sensitive and robust proteomic methods, including associated software for the analysis and characterisation of these complexes is a major priority.
(ii)Proteomics community (both academic and industrial): There are already close collaborations between the University of Cambridge and several mass spectrometry vendors and proteomics software vendors including Thermo Finnigan, Applied Biosystems, Waters and Matrix Science. The proteomics community as a whole will benefit from the development of such widely applicable methodology and associated software. KSL has collaborated with Matrix Science and Applied Biosystems for many years including Applied Biosystems contribution to a BBSRC award which resulted in the first developments of organelle proteomics methods. We envisage that such collaborations with mass spectrometry vendors will continue throughout the period of this grant, and will result in us working with the appropriate vendors to incorporate them into the workflow.
(iii)Pharmaceutical industry: The APJ and KSL have given talks frequently to the pharmaceutical industry on cell biology, ion channels and proteomics, including Glaxo Smith Kline and Pfizer. We have numerous contacts with these industrial research groups, including the newly established Pfizer research groups at Granta Park Cambridge. KSL has discussed proteomic methodology with Glaxo Smith Kline, Astra Zeneca and also Genentech. The methodologies developed within the context of this proposal are so fundamental that they could underpin many area of pharmaceutical research.
(iv)Full descriptions of this work will be published in peer reviewed literature and presented at academic conferences.
(v) Software developed as part of this project will be freely disseminated to the scientific community.
Publications
Salvage SC
(2021)
Ca2+-dependent modulation of voltage-gated myocyte sodium channels.
in Biochemical Society transactions
Salvage S
(2021)
Ca2+-dependent modulation of voltage-gated myocyte sodium channels.
Rees JS
(2015)
Protein Neighbors and Proximity Proteomics.
in Molecular & cellular proteomics : MCP
Rees JS
(2017)
Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters.
in Current protocols in protein science
Rees JS
(2015)
Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters.
in Current protocols in protein science
Rees JS
(2020)
Identification of the cis-molecular neighbours of the immune checkpoint protein B7-H4 in the breast cancer cell-line SK-BR-3 by proteomic proximity labelling.
in International journal of oncology
Queiroz RML
(2022)
Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.
in The Biochemical journal
Li XW
(2014)
New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.
in The Journal of biological chemistry
Description | Many of the functionally critical protein-protein interactions that occur on the surface of cells are of low affinity. Some of these protein complexes are internalised together into cells and interact closely with other proteins as they move through the intracellular trafficking pathways. These low affinity interactions make their co-isolation, and hence their subsequent characterisation difficult using conventional mass spectrometry approaches. The short Tools and Resources grant was designed to develop and extend a protocol we have developed (SPPLAT) as outlined in the grant. Key findings: 1) we have successfully established SPPLAT as a sensitive and quantitative proteomic method for the analysis of protein clusters on the plasma membrane. 2) We have identified several novel interactors with the chicken B cell receptor on DT40 lymphocyte cells. 3) We have shown that several of these regulate integrin signalling willowing B cell receptor activation. 4) We have extended the SPPLAT methodology to cover dynamic aspects of proteins that interact with differing partners as they traverse the endocytic pathway. 5)We have identified at least one novel partner for the transferrin receptor within the endocytic compartment. Final analysis of the dynamic SPPLAT data set is currently being undertaken. Additional confirmatory experiments will be required. |
Exploitation Route | There is potential for the technique as a general tool to examine proteins in localised plasma membrane clusters. We are collaborating with groups in industry to apply this methodology to current problems in cell biology and immunology. We have begun collaborative projects with Medimmune (Cambridge UK), to apply this method to identify proteins clustering with the IL7 receptor on human T cells and neighbours (both in cis and trans) with the B7 protein family of immune regulators. We are currently exploring the potential for SPPLAT to examine neighbour proteins of the voltage-gated sodium channel on neurones. |
Sectors | Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
URL | http://www.bioc.cam.ac.uk/people/uto/jacksont |
Description | This work established a method for the analysis of local protein clusters on the plasma membrane of cells and examined the potential of the technique to study the changing composition of these protein clusters during a simple membrane trafficking pathway (endocytosis). Application of the method to the 'fostering global economic performance, and specifically the economic competitiveness of the United Kingdom, increasing the effectiveness of public services and policy,enhancing quality of life' in the sense that the method enables an improved analysis of membrane protein assembles, many of which (eg ion channels, immune system proteins) are of potential therapeutic importance. We are now collaborating with Medimmune and Genetech to apply this method to specific biomedical problems. |
Sector | Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Cultural |
Description | applications of a novel proteomic proximity labelling assay to identify membrane proteins of therapeutic interest |
Amount | £104,856 (GBP) |
Organisation | AstraZeneca |
Department | MedImmune |
Sector | Private |
Country | United Kingdom |
Start | 08/2015 |
End | 02/2017 |
Description | identification ofIL7/TCR interactome using MS based proteomics and relationship to T cell fate and function |
Amount | £129,241 (GBP) |
Organisation | AstraZeneca |
Department | MedImmune |
Sector | Private |
Country | United Kingdom |
Start | 04/2015 |
End | 04/2017 |
Title | Selective proteomic proximity labelling assay using tyramide (SPPLAT) |
Description | A method for the quantitative analysis of protein near-neighbours within localised protein clusters on membrane surfaces. |
Type Of Material | Technology assay or reagent |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | A paper describing the method has been published (Li et al ). A detailed method protocol has been published in Current protocols in Protein Science. A review on the method and its similarities and differences to other proximity labelling methods has been published in Molecular and Cellular Proteomics(see publications). We have been approached by several groups in both industry and academia with the aim of establishing collaborative projects applying the method to distinct biological and biomedical problems that require the application of quantitative proteomics. We are currently collaborating with researchers at Medimmune to use the method to examine and detect both cis and trans-protein neighbours of immune signalling molecules. |
URL | http://www.cam.ac.uk/research/news/neighbourhood-watch-new-technique-helps-identify-proteins-involve... |
Description | A new proteomic method to investigate localised surface protein clusters. |
Organisation | Chinese Academy of Sciences |
Department | Institute of Biophysics |
Country | China |
Sector | Academic/University |
PI Contribution | Many proteins, including the sodium channels exist on the plasma membrane as localised protein clusters in which individual proteins interact weakly. These proteins are likely to influence each other's functions, but their identification by classical immunopreciptation and mass spectrometry (MS) identification is currently difficult, because the low affinity interactions fall apart during immunoprecipitation. We have developed a novel MS method we call 'selective proteomic proximity labelling using tyramide (SPPLAT). The method relies on the fact that peroxidase converts tyramide into a very unstable radical that either quenches or covalently labels proteins within a few dozen nm. The method involves targeting peroxidase to a protein of interest within a surface protein cluster via an antibody. Then the cells are briefly incubated with a tyramide-biotin conjugate. Proteins in the immediate vicinity of the target are covalently biotinylated and can therefore be isolated by streptavidin pull-down and idientified by MS. |
Collaborator Contribution | The work described above was carried out in both Beijing and Cambridge by an IBP student under my supervision and the supervision of Prof Perrett |
Impact | A paper describing SPPLAT:http://www.ncbi.nlm.nih.gov/pubmed/24706754 Oral presentation of the method by myself and Dr Rees. |
Start Year | 2010 |
Description | applications of novel proteomic proximity labelling assay to identify membrane proteins of therapeutic interest |
Organisation | AstraZeneca |
Department | MedImmune |
Country | United Kingdom |
Sector | Private |
PI Contribution | We are collaborating with the Institute of Biophysics, Beijing in the development of SPPLAT technology As a result of this work we have initiated a collaboration with Medimmune (Astra Zeneca) (Cambridge) to develop SPPLAT on immune cells. Two such projects are now being undertaken. One examines the neighbours of the T cell receptor in interleukin-stimulated T cells. The other project is using the method to examine both cis and trans protein neighbours of the B7 family of cell-adhesion molecules implicated in the immune-destruction of cancer cells. These are projects whose funding was directly inspired by our original work on the method described in the grant. |
Collaborator Contribution | Work on the SPPLAT method development was carried out jointly in Beijing and Cambridge by JS Rees (Cambridge) and Li Xue-we (Institute of Biophysics, Beijing). |
Impact | The method, a detailed protocol and a review on the method have been published (see publications). |
Start Year | 2010 |
Description | A new proteomic method to study local protein surface clusters |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | A press release to describe the general importance to cell biology and proteomics of our new proteomic method. Contact from several pharmaceutical companies to apply SPPLAT to specific problems such as ion channels on pain neurones, and signalling molecules on immune cells. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.cam.ac.uk/research/news/neighbourhood-watch-new-technique-helps-identify-proteins-involve... |
Description | poster presentation and invited speaker at EMBO workshop on proximity assays |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | A poster presentation on SPPLAT (including the use to study endocytic pathways) to industrial and academic groups. JSRR presented a poster on our SPPLAT methodology and described the method in an invited oral presentation (as a young investigator presentation). We received further interest from academic groups with a view to further collaborations |
Year(s) Of Engagement Activity | 2014 |
URL | http://events.embo.org/14-pla/index.html |
Description | publicity notice |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Generated wide interest from many industrial and academic research groups As a result, we are now pursing potential industrial and academic collaborations including Astrazeneca, Pfizer and the BBSRC institute of animal health Purbright. |
Year(s) Of Engagement Activity | 2014 |
URL | http://www.cam.ac.uk/research/news/neighbourhood-watch-new-technique-helps-identify-proteins-involve... |