An optimized proteomics-based approach to estimate blood contamination and cellular heterogeneity of frozen placental tissue

Archived human placental tissue specimens are vital for studying placenta pathophysiology and toxicology. Proteomics analysis of placental tissue provides mechanistic and translational information, but the highly perfused and heterogenous nature of the placenta creates confounding technical variability. In this study, we developed an optimized proteomics-based approach to address the technical variability of proteomics data by normalizing blood contamination and cellular heterogeneity of archived placenta samples. Placenta samples (n = 99) were homogenized, digested using trypsin, and analyzed by liquid chromatography mass-spectrometry. Label-free quantification (LFQ) intensities of the proteins were analyzed for their correlation with blood (albumin) and placenta (aromatase) markers. Proteins that positively correlated with albumin and negatively correlated with aromatase or vice versa were considered blood and placental proteins, respectively. Next, the cellular heterogeneity of individual placenta samples was evaluated by quantifying specific cellular markers of cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, fibroblasts, Hofbauer cells, and decidual cells. We found that placental proteins were contaminated by 41 to 85% blood proteins. Analysis of cellular markers confirmed syncytiotrophoblasts as the major cell type in placenta (i.e., 41 ± 9% of all cell types). Two samples showed distinct cell compositions with higher levels of the extravillous trophoblasts and decidual cells. In summary, the optimized proteomics-based approach to estimate blood contamination and cellular heterogeneity of placental tissues has the potential to address technical variability in placenta proteomics analysis, which can be extended to other highly perfused and heterogenous tissues.