(1072-A) Increasing protein extraction throughput for cell-based assays; integration of an AFA-assisted ultrasonication and an automated liquid handling workstation to study biological perturbations
Monday, February 5, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: Drug screening and identification of potential biomarkers and therapeutic targets requires a rapid process of routine protein quantification, where biological interactions in perturbed cells can be evaluated to enable the study of specific mechanisms. Such a high-throughput (HTP) system can allow an individual’s proteome to be determined and enable the evaluation of their risk to different diseases, a diagnostic approach towards precision medicine via molecular phenotyping. We have previously developed a HTP cell-based assay, via a bottom-up proteomics approach to investigate biological perturbations and here, we have automated many aspects of the protein extraction workflow, showcasing the process to yield a higher throughput and deeper proteome coverage as well as displaying significant implications from their biological perturbations. AC16 human cardiomyocytes (Sigma) were cultured in a 96-well plate and optimized density of cells with/without drug were transferred in a detergent-free buffer onto a Covaris TPX 150 plate for Adaptive Focused Acoustics (AFA)-assisted ultrasonication on an LE220-plus instrument (Covaris) after a series of liquid transfer steps in a humidity-controlled Biomek i7 (Beckman) automated workstation. Proteins were subsequently reduced, alkylated, and trypsin digested (1:20 µg ratio of trypsin/protein), after which desalted peptides were loaded on an Ultimate 3000 (Thermo) liquid chromatography system coupled to an Exploris 480 Orbitrap (Thermo) mass spectrometer. Data-Independent Acquisition (DIA) was performed over a 45-minute gradient and searched using the Pan human library (PXD000954) with a statistically significant false discovery rate reference. Our automated protein extraction workflow yielded approximately 0.6 µg/µl (n=3, CV=5.72) of protein concentration for a total amount of 10 ug from ~ 40,000 cells grown on a 96-well plate following ultrasonication at representative AFA settings (350 PIP, 50 DF and 200 CPB). The comparable protein yield to our previous manual approach of harvesting cells from a 6-well plate enabled the assessment of FCCP drug concentrations on cardiomyocytes, a potent uncoupler of oxidative phosphorylation in mitochondria that disrupts ATP synthesis. Data analysis allowed the quantification of approximately 3,000 proteins over 7 orders of magnitude dynamic range, of which 50 were identified to impact cellular amino acid metabolic processes. Differential expression analysis of cells treated with FCCP also displayed the upregulation of the DNA mismatch repair protein Msh3 as well as several other proteins involved in DNA transcription modulation. With our fully automated HTP cell-based assay system, cells can be directly harvested on a 96-well plate, allowing the investigation of multiple conditions for drug screening studies, without sacrificing throughput and proteomic coverage. We hope to adapt this workflow on both label free and stable isotope-labeling to allow for the study of post-translational modifications and help identify important key regulators in cell-based assays. We envision that this platform would ultimately allow cellular perturbations to be rapidly studied and investigated.