Protein Profiling of Primary Human Samples for Pathway Analysis and Patient Stratification

Abstract

In recent years, scientific progress has led to the advancement of precision medicine, a novel therapeutic strategy. For this purpose, molecular investigation of patient-derived samples is a necessity. Genetic analysis has been the gold standard in this field for years. However, in a cell, proteins and their posttranslational modifications lead to differences in genotype and phenotype. Therefore, the use of genetic information as basis for treatment decisions does not always translate into therapeutic benefits. The integration of proteomic approaches to further elucidate pathophysiological mechanisms is essential. Protein analysis methods need to be flexible to be used in different sample types and provide high sensitivity as well as throughput to complement these novel therapeutic approaches. The recently emerging DigiWest technology allows for detection of numerous proteins and protein variants from a single sample. Here, the DigiWest workflow was adapted and modified for protein analysis from clinically relevant sample types, such as formalin-fixed or fresh frozen tissue extracts and blood samples. A novel serum-screening platform was designed and established. Through the integration of authentic antigens, the parallel detection of immunoglobulins against different pathogenic strains of coronaviruses was achieved. Furthermore, multiplexed protein analysis from minuscule formalin-fixed and paraffin-embedded cervical punch biopsies by DigiWest was established. In vivo treatment effects of non-invasive-physical plasma, a novel therapeutic approach for treatment of cervical intraepithelial neoplasia, were evaluated and upon comparing results to in vitro cell culture experiments, general trends in treatment response could be confirmed. However, differences in protein expression profiles emphasize the need for molecular investigation of treatment effects in vivo. The potential of protein analysis of fresh frozen samples was explored by referencing snap frozen breast cancer biopsies. The multiplexed detection of several infiltrating immune cell markers by DigiWest as prognostic factor was established. A subset of co-expressed immune cell markers, revealing a positive influence on patient outcome was identified and induced changes in several pathways were detected. Overall, sample preparation, assay strategies and analysis tools were adapted to the multiplexed protein analysis of different human sample types via DigiWest and the unique potential of this approach was demonstrated.