Universal Solid-Phase Protein Preparation (USP3) for Bottom-up and Top-down Proteomics.

Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP3) method which is rapid, robust, and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis. This technique builds upon the single-pot solid-phase-enhanced sample preparation (SP3) where we now demonstrate its scalability (low to high micrograms of protein) and the influence of variables such as bead and enzyme amounts on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extraction resulting in a more reliable method that is simple and easy to implement for routine and high-throughput analysis of proteins. We benchmarked the performance of this technique against filter-aided sample preparation (FASP) using 30 µg of total HeLa protein lysate. We also show that the USP3 method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 µg of HeLa protein lysate. The USP3 protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and analysis by MS.

S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer.

Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFß1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.