Proteomic profiling of preeclampsia

Preeclampsia (PE) occurs in about 5% of all pregnancies. It is a complex disease characterized by sudden-onset hypertension, and it is harmful to the mother and the growing baby, accounting for 40% of fetal deaths worldwide. The overall objective of this project is to identify molecular subtypes of PE and to further apply these subtypes to liquid biopsies, including plasma and isolated placental syncytiotrophoblast-derived extracellular vesicles, enabling the identification of biomarker panels for PE prediction and early diagnosis.

For tissue proteomics, a pilot cohort of 24 placentas (eight early- and eight late-onset PE and eight controls) was processed according to our published method (Kuras et al., 2018). Samples were acquired using two protocols: a more standardized one and one with longer processing times and unspecified collection sites. Proteomic analysis was performed using DIA–PASEF on an Evosep LC coupled to a timsTOF HT mass spectrometer. Following off-line fractionation of pooled placenta samples, a spectral library was built in FragPipe, and the raw data was searched using DIA-NN. Further analyses were performed in Perseus and RStudio. Extracellular vesicles were isolated on an acoustic trapping platform, described in detail previously (Broman et al., 2021). Using the acoustic trap, extracellular vesicles (EVs) were effectively isolated from 10 ml of plasma.

A project-specific spectral library consisting of 9,917 proteins was built, of which 7,202 proteins were quantified in the 24 samples, 6,700–7,000 per sample. Sample collection and storage-related alterations in proteome integrity were revealed in the data analysis. We observed a significantly lower level of mitochondrial and ribosomal proteins in the compromised samples. In contrast, acute and chronic inflammation and hemoglobin protein chains showed higher expression, emphasizing the importance of the chain of custody during sample collection and storage for reliable protein quantification. When investigating the samples collected in a standardized manner (stored at 4°C and processed within 4 h), early-onset PE differed more from late-onset PE and controls and displayed upregulation of proteins related to inflammation, hypoxia, and metabolism. In contrast, extracellular matrix and translation-related proteins showed lower expression levels in early-onset PE.

A workflow enabling high-throughput, large-scale proteomic characterization of placenta tissue was developed and successfully applied to a small cohort in which we, to the best of our knowledge, identified and quantified more proteins compared to previously published studies. Moreover, this study provides insight into the importance of sample collection and storage. Additionally, the successful isolation of EVs from minute plasma volumes enables the utilization of large cohorts of biobank samples.