Background & Aims
High-throughput sequencing (“-omics”), have revolutionised the identification of markers and pathways in pain. Even so, the identification of pain-related mechanisms remains challenging due to the complex nature of pain pathophysiology, data sparsity, and species differences across tissues. Crucially, the focus on transcriptomics also overlooks the functional building blocks of the cells – the proteins. While some correlation is seen, resulting landscapes are known to be different [1].
Here, we expand the available -omics landscape for human dorsal root ganglia (DRG) to include quantitative proteomics via data-independent acquisition coupled with parallel accumulation serial fragmentation (DIA-PASEF). Through multi-omic and cross-species analyses, we aim to establish a state-of-the-art proteome of the human DRG. This allows for an in-depth comparison of the protein landscape to previously published transcriptomic and epigenomic studies, and lays foundation for subsequent work.
Methods
Lumbar human DRG were collected from male and female donors within 4 hours of cross-clamp. Proteins were extracted as previously described for other tissues [2] for analysis on a timsTOF HT mass spectrometer in DIA-PASEF mode. Raw spectra were processed with DIA-NN [3] followed by subsequent processing in R and python. Cross-species and multi-omic data integration across relevant studies were performed using a combination of supervised PCA, gene set enrichment analyses, and probabilistic classification through sklearn. Cell-type deconvolution was performed with CARD and Redeconv, using snRNA-seq and spatial-seq datasets as reference.
Results
Using quantitative proteomics, we have studied male and female lumbar DRG from healthy human donors. DIA-PASEF provides the most comprehensive proteome coverage available to date: This gives deep proteome coverage to explore sex differences, multi-omic comparisons, and cross-species comparisons. Here, we describe the similarities and differences between bulk proteomics and bulk transcriptomics, while highlighting the relationship of sex and proteome versus sex and transcriptomics within human DRGs [4]. Building on this, the availability of single nuclear and spatial-seq datasets [5,6] allows for cell type deconvolution of bulk data to extract neuronal and immune signatures for further sex analyses.
This work is complemented by comparisons to mouse DRG proteomic data. Given the reliance of mouse models in the study of pain, detailing correlations at the proteome level may improve the translatability of these studies.
Conclusions
Together, these data contribute to the detailed phenotyping of male and female human DRG and provide novel insights into cross-species and cross-omic relationships within lumbar ganglia.
References
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[5] Nguyen, M. Q., von Buchholtz, L. J., Reker, A. N., Ryba, N. J. P., & Davidson, S. (2021). Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons. ELife. https://doi.org/10.7554/eLife.71752
[4] Ray, P. R., Shiers, S., Caruso, J. P., Tavares-Ferreira, D., Sankaranarayanan, I., Uhelski, M. L., Li, Y., North, R. Y., Tatsui, C., Dussor, G., Burton, M. D., Dougherty, P. M., & Price, T. J. (2022). RNA profiling of human dorsal root ganglia reveals sex differences in mechanisms promoting neuropathic pain. Brain. https://doi.org/10.1093/brain/awac266
[6] Tavares-Ferreira, D., Shiers, S., Ray, P. R., Wangzhou, A., Jeevakumar, V., Sankaranarayanan, I., Cervantes, A. M., Reese, J. C., Chamessian, A., Copits, B. A., Dougherty, P. M., Gereau, R. W., Burton, M. D., Dussor, G., & Price, T. J. (2022). Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors. Science Translational Medicine, 14(632), eabj8186.
[1] Wang, D., Eraslan, B., Wieland, T., Hallström, B., Hopf, T., Zolg, D. P., Zecha, J., Asplund, A., Li, L., Meng, C., Frejno, M., Schmidt, T., Schnatbaum, K., Wilhelm, M., Ponten, F., Uhlen, M., Gagneur, J., Hahne, H., & Kuster, B. (2019). A deep proteome and transcriptome abundance atlas of 29 healthy human tissues. Molecular Systems Biology, 15(2). https://doi.org/10.15252/msb.20188503
[2] Xian, F., Sondermann, J. R., Varela, D. G., & Schmidt, M. (2022). Deep proteome profiling reveals signatures of age and sex differences in paw skin and sciatic nerve of naïve mice. ELife, 11. https://doi.org/10.7554/eLife.81431