Transcriptomics and Spatial Proteomics for Discovery and Validation of Missing Proteins in the Human Ovary

Abstract

Efforts to understand the complexities of human biology encompass multidimensional aspects, with proteins emerging as crucial components. However, studying the human ovary introduces unique challenges due to its complex dynamics and changes over a lifetime, varied cellular composition, and limited sample access. Here, four new RNA-seq samples of ovarian cortex spanning ages of 7 to 32 were sequenced and added to the existing data in the Human Protein Atlas (HPA) database , opening the doors to unique possibilities for exploration of oocyte-specific proteins. Based on transcriptomics analysis of the four new tissue samples representing both prepubertal girls and women of fertile age, we selected 20 protein candidates that lacked previous evidence at the protein level, so-called “missing proteins” (MPs). The proteins were validated using high-resolution antibody-based profiling and single-cell transcriptomics. Fourteen proteins exhibited consistent single-cell expression patterns in oocytes and granulosa cells, confirming their presence in the ovary and suggesting that these proteins play important roles in ovarian function, thus proposing that these 14 proteins should no longer be classified as MPs. This research significantly advances the understanding of MPs, unearthing fresh avenues for prospective exploration. By integrating innovative methodologies and leveraging the wealth of data in the HPA database, these insights contribute to refining our understanding of protein roles within the human ovary and opening the doors for further investigations into missing proteins and human reproduction.

The 2023 Report on the Proteome from the HUPO Human Proteome Project

“…One such example is extended analysis of ovarian samples from women of fertile age, that allowed for localizing multiple proteins defined as “missing proteins” to various structures of follicle cells . As a follow-up to this study, four new ovary RNA samples have been sequenced, including samples from prepubertal women and young adults, from whom oocytes were captured and analyzed, as well as granulosa, stromal, and follicular cells . This paper is an outstanding example of biology-driven studies yielding several previously missing proteins.…”

“…The U.S. National Cancer Institute’s Clinical Proteomics Tumor Analysis Consortium (CPTAC) and International Cancer Proteomics Consortium (ICPC) have set standards, established procedures, provided reagents, and populated massive data sets on more than a dozen major types of cancers (see Cancer B-D/HPP report). Proteomics has also advanced insights into COVID-19, ,, cardiovascular, , and neurological disorders, and diseases of the eye. New workflows are being developed for working with low-level ,,− and low-volume samples. , Network analysis continues to inform studies on disease-related pathways. − …”