class: center, middle, inverse, title-slide # Proteomic and bioinformatics analyses of plasma from SCI neurological improvers and non-improvers ### Gabriel Mateus Bernardo Harrington ### Keele University ### ISCoS 2021: VIRTUAL<br>Presented: 2021-10-01<br>(updated: 2021-09-08) --- ## Speaker declaration I declare to have no interests in the below: - The existence of any significant financial activity or other relationship - Financial or material compensation in relation to research and publishing - Financial or material compensation in relation to educational activities - Ownerships and possessions in companies related to health care (includes service provides, IT) - Compensation for expert functions in health care and consulting health care guidance processes .center[ <div id="htmlwidget-7f4ee0390d75fc0ff32e" style="width:756px;height:288px;" class="leaflet html-widget"></div> <script type="application/json" data-for="htmlwidget-7f4ee0390d75fc0ff32e">{"x":{"options":{"crs":{"crsClass":"L.CRS.EPSG3857","code":null,"proj4def":null,"projectedBounds":null,"options":{}}},"calls":[{"method":"addTiles","args":["//{s}.tile.openstreetmap.org/{z}/{x}/{y}.png",null,null,{"minZoom":0,"maxZoom":18,"tileSize":256,"subdomains":"abc","errorTileUrl":"","tms":false,"noWrap":false,"zoomOffset":0,"zoomReverse":false,"opacity":1,"zIndex":1,"detectRetina":false,"attribution":"© <a href=\"http://openstreetmap.org\">OpenStreetMap<\/a> contributors, <a href=\"http://creativecommons.org/licenses/by-sa/2.0/\">CC-BY-SA<\/a>"}]}],"setView":[[52.884422,-3.033414],16,[]]},"evals":[],"jsHooks":[]}</script> ] ??? - What an odd compulsory speil... --- ## Prior work - Modelling of SCI neurological outcomes with routine haematological markers <a name=cite-brown_preliminary_2019></a><a name=cite-bernardo_harrington_routinely_2020></a>([Brown, Harrington, Hulme, Morris, Bennett, Tsang, Osman, Chowdhury, Kumar, and Wright, 2019](https://doi.org/10.1089/neu.2019.6495); [Bernardo Harrington, Cool, Hulme, Osman, Chowdhury, Kumar, Budithi, and Wright, 2020](https://doi.org/10.1089/neu.2020.7144)) - Found markers associated with liver function added modest predictive value - Implicates liver status/metabolic health with neurological outcomes .center[ <img src="index_files/figure-html/rsquare-plot-1.png" width="50%" /> ] --- ## "Bottom-up" Proteomics - A brief overview 1. Extract consistent total protein concentration for each sample 1. Optional dimensionality reduction steps (e.g. via Proteominer™ beads) 1. Digest proteins (Trypsin is commonly used) 1. For labelled techniques, the labels are applied 1. Samples are fed through some flavour of chromatography and into the mass spec (e.g. high-performance liquid chromatography) -- ### Techniques used: - Isobaric tag for relative and absolute quantitation ([iTRAQ](https://en.wikipedia.org/wiki/Isobaric_tag_for_relative_and_absolute_quantitation)) and [label-free](https://en.wikipedia.org/wiki/Label-free_quantification) proteomic experiments were conducted .center[ <img src="../proteomic_talk_2020-09-24/itraq_labels.png" width="40%" /> ] ??? - Highlight complexity and uncertainty in measurements - put data in context, further work needed etc. --- ## Dynamic range reduction via<br>Proteominer™ beads - Abundant proteins saturate beads, whereas less abundant proteins don't - Once the column is washed, the excess abundant proteins are removed, and the less abundant proteins are retained, thus the dynamic range is reduced <img src="label-free_and_itraq_image_ch.png" width="699" style="display: block; margin: auto;" /> --- ## Experimental design - Plasma from SCI human patients - Two time points: 1. "Acute": ~2-weeks post-injury 1. "Subacute": ~3-months post-injury<sup>*</sup> - Improvers are AIS C patients who experienced an AIS grade conversion - 2 4-plex iTRAQ runs and a more recent label-free experiment with all samples <table class="table table-hover" style="margin-left: auto; margin-right: auto;"> <thead> <tr> <th style="text-align:left;"> Initial AIS grade </th> <th style="text-align:left;"> Improver </th> <th style="text-align:right;"> “Acute” </th> <th style="text-align:right;"> “Subacute” </th> </tr> </thead> <tbody> <tr> <td style="text-align:left;"> A </td> <td style="text-align:left;"> </td> <td style="text-align:right;"> 11 </td> <td style="text-align:right;"> 9 </td> </tr> <tr> <td style="text-align:left;"> C </td> <td style="text-align:left;"> Yes </td> <td style="text-align:right;"> 10 </td> <td style="text-align:right;"> 9 </td> </tr> <tr> <td style="text-align:left;"> C </td> <td style="text-align:left;"> No </td> <td style="text-align:right;"> 7 </td> <td style="text-align:right;"> 6 </td> </tr> <tr> <td style="text-align:left;"> D </td> <td style="text-align:left;"> </td> <td style="text-align:right;"> 11 </td> <td style="text-align:right;"> 10 </td> </tr> </tbody> </table> .footnote[<sup>*</sup> iTRAQ experiments did not include AIS A or D groups at the subacute time point] --- ## iTRAQ key pathway - Complement cascade - Total number of proteins identified: 79 - Majority of proteins identified are part of the complement cascade, differences in these pathways appears to be at least partially responsible for heterogeneous outcomes - Many of these proteins interact with the liver in some capacity <img src="hsa04610_pathview.png" width="586" style="display: block; margin: auto;" /> ??? The fold changes in the KEGG pathway are from the Acute AIS C improvers relative to non-improvers --- ## iTRAQ key protein identified: Serum amyloid A1 .pull-left[ <img src="index_files/figure-html/saa1-plot-1.png" width="504" /> ] .pull-right[ - [Serum amyloid A1](https://www.uniprot.org/uniprot/P0DJI8) - Precursor of amyloid A, the aberrant deposition of which leads to inflammatory amyloidosis <a name=cite-sun_serum_2016></a>([Sun and Ye, 2016](https://doi.org/10.1016/j.gene.2016.02.044)) - Produced by hepatocytes, during acute phase response they associate with HDL, displacing ApoA1 <a name=cite-benditt_amyloid_1977></a>([Benditt and Eriksen, 1977](https://doi.org/10.1073/pnas.74.9.4025)) - SAA binding HDL is thought to favour removal of cholesterol from sites of inflammation - SAA can also bind retinol, potentially limiting bacterial burden in tissues <a name=cite-derebe_serum_2014></a>([Derebe, Zlatkov, Gattu, Ruhn, Vaishnava, Diehl, MacMillan, Williams, and Hooper, 2014](https://doi.org/10.7554/elife.03206)) ] ??? ### Proteinpilot - way more abundant in subacute C imp vs non-imp (~28 fc) - even more so in acute C imp vs D ### Openms - Downreg in acute C imp vs non-imp (~ -2.5 fc) - Upreg in subacute C imp vs non-imp (~ 2.4 fc) - Downreg in C groups and D vs A --- ## Label-free proteomics - Data still being analysed - Substantial overlap in proteins identified - Complement cascade still the most significant - Early proteins of interest: - Peroxiredoxin 2, detected in Acute C improvers but not Acute C non-improvers. Involved in oxidative stress response - Several immunoglobulins variable and constant regions, potential link to work suggesting IVIG therapy benefiting outcomes? <a name=cite-brennan_ivig_2016></a>([Brennan, Kurniawan, Vukovic, Bartlett, Käsermann, Arumugam, Basta, and Ruitenberg, 2016](https://doi.org/10.1002/acn3.318)) ### Link between metabolic status and SCI? - Mounting evidence to support a link between the liver, and perhaps metabolic health more broadly, and SCI, including neurological outcomes - Excellent review by Goodus and McTigue on the topic <a name=cite-goodus_hepatic_2020></a>([Goodus and McTigue, 2020](https://doi.org/10.1016/j.expneurol.2019.113160)) ??? - touch on future work - validation at Glasgow --- ## Links to this slide deck: The rendered slides can be found at this address: - [https://h-mateus.github.io/presentations/iscos_sci_proteomics_2021-08-31/index.html#1](https://h-mateus.github.io/presentations/iscos_sci_proteomics_2021-08-31/index.html#1) .center[ <img src="slides_qr-code.png" width="40%" /> The raw files are on GitHub [here](https://github.com/H-Mateus/presentations) under the GPL license ] --- ## Acknowledgements .pull-left[ - Thanks to my supervisors: Karina Wright, Paul Cool, Charlotte Hulme & the rest of the [OsKOR team](https://oskor.netlify.app/) - Thanks to the clinical team at the [Midland Centre for Spinal Injuries](https://www.rjah.nhs.uk/Our-Services/Spinal-Injuries-Unit.aspx), and all the patients who've donated to our biobank - Thanks for the funding: [EPSRC](https://epsrc.ukri.org/) - Thanks to the open-source community for facilitating this research, Yihui Xie's R package [xaringan](https://github.com/yihui/xaringan) in particular for these slides ] .pull-right[ <img src="../logos/EPSRC+logo.png" width="90%" /><img src="../logos/keele_logo.jpg" width="90%" /><img src="../logos/rjah_logo.png" width="90%" /> ] --- ## References <a name=bib-benditt_amyloid_1977></a>[Benditt, E. P. and N. Eriksen](#cite-benditt_amyloid_1977) (1977). "Amyloid Protein SAA Is Associated with High Density Lipoprotein from Human Serum". En. In: _Proceedings of the National Academy of Sciences_ 74.9, pp. 4025-4028. ISSN: 0027-8424, 1091-6490. DOI: [10.1073/pnas.74.9.4025](https://doi.org/10.1073%2Fpnas.74.9.4025). <a name=bib-bernardo_harrington_routinely_2020></a>[Bernardo Harrington, G. M., P. Cool, C. Hulme, et al.](#cite-bernardo_harrington_routinely_2020) (2020). "Routinely Measured Haematological Markers Can Help to Predict AIS Scores Following Spinal Cord Injury". In: _Journal of Neurotrauma_. ISSN: 0897-7151. DOI: [10.1089/neu.2020.7144](https://doi.org/10.1089%2Fneu.2020.7144). <a name=bib-brennan_ivig_2016></a>[Brennan, F. H., N. D. Kurniawan, J. Vukovic, et al.](#cite-brennan_ivig_2016) (2016). "IVIg Attenuates Complement and Improves Spinal Cord Injury Outcomes in Mice". En. In: _Annals of Clinical and Translational Neurology_ 3.7, pp. 495-511. ISSN: 2328-9503. DOI: [10.1002/acn3.318](https://doi.org/10.1002%2Facn3.318). <a name=bib-brown_preliminary_2019></a>[Brown, S. J., G. M. Harrington, C. H. Hulme, et al.](#cite-brown_preliminary_2019) (2019). "A Preliminary Cohort Study Assessing Routine Blood Analyte Levels and Neurological Outcome after Spinal Cord Injury". In: _Journal of Neurotrauma_. ISSN: 0897-7151. DOI: [10.1089/neu.2019.6495](https://doi.org/10.1089%2Fneu.2019.6495). --- ## References cont. <a name=bib-derebe_serum_2014></a>[Derebe, M. G., C. M. Zlatkov, S. Gattu, et al.](#cite-derebe_serum_2014) (2014). "Serum Amyloid A Is a Retinol Binding Protein That Transports Retinol during Bacterial Infection". In: _eLife_ 3. Ed. by F. M. Powrie, p. e03206. ISSN: 2050-084X. DOI: [10.7554/elife.03206](https://doi.org/10.7554%2Felife.03206). <a name=bib-goodus_hepatic_2020></a>[Goodus, M. T. and D. M. McTigue](#cite-goodus_hepatic_2020) (2020). "Hepatic Dysfunction after Spinal Cord Injury: A Vicious Cycle of Central and Peripheral Pathology?" En. In: _Experimental Neurology_ 325, p. 113160. ISSN: 0014-4886. DOI: [10.1016/j.expneurol.2019.113160](https://doi.org/10.1016%2Fj.expneurol.2019.113160). <a name=bib-sun_serum_2016></a>[Sun, L. and R. D. Ye](#cite-sun_serum_2016) (2016). "Serum Amyloid A1: Structure, Function and Gene Polymorphism". En. In: _Gene_ 583.1, pp. 48-57. ISSN: 0378-1119. DOI: [10.1016/j.gene.2016.02.044](https://doi.org/10.1016%2Fj.gene.2016.02.044).