Spatial Proteomics for Further Exploration of Missing Proteins: A Case Study of the Ovary

In the quest for "missing proteins" (MPs), the proteins encoded by the human genome still lacking evidence of existence at the protein level, novel approaches are needed to detect this challenging group of proteins. The current count stands at 1,343 MPs, and it is likely that many of these proteins are expressed at low levels, in rare cell or tissue types, or the cells in which they are expressed may only represent a small minority of the tissue. Here, we used an integrated omics approach to identify and explore MPs in human ovaries. By taking advantage of publicly available transcriptomics and antibody-based proteomics data in the Human Protein Atlas (HPA), we selected 18 candidates for further immunohistochemical analysis using an exclusive collection of ovarian tissues from women and patients of reproductive age. The results were compared with data from single-cell mRNA sequencing, and seven proteins (CTXN1, MRO, RERGL, TTLL3, TRIM61, TRIM73, and ZNF793) could be validated at the single-cell type level with both methods. We present for the first time the cell type-specific spatial localization of 18 MPs in human ovarian follicles, thereby showcasing the utility of the HPA database as an important resource for identification of MPs suitable for exploration in specialized tissue samples. The results constitute a starting point for further quantitative and qualitative analysis of the human ovaries, and the novel data for the seven proteins that were validated with both methods should be considered as evidence of existence of these proteins in human ovary.