High-Throughput Workflow for Detergent-free Cell-Based Proteomic Characterization

We have developed an automated cell-based workflow for the quantification of proteins by liquid chromatography–mass spectrometry (LC-MS) that facilitates large-scale perturbation studies carried out in a 96-well plate format and enables the preparation of one full plate in approximately 4 h, showcasing a high-throughput (HTP) concept. Cells were grown in a 96-well plate and lysed via ultrasonication. Proteins were subsequently solubilized, extracted, and processed into tryptic peptides for 2 h before being acquired by data-independent acquisition mass spectrometry (DIA-MS). This workflow leverages adaptive focused acoustics (AFA) technology for ultrasonication to aid cell lysis and protein solubilization on an automated liquid handling platform. As proof of principle, AC16 human cardiomyocyte-like cells were cultured in a 96-well plate under optimized conditions that were compatible with the downstream HTP pipeline. Over 30,000 peptides were identified, corresponding to the detection of 5100 unique proteins. 50% of measured proteins had an average coefficient of variation (CV) under 25% from approximately 30,000 cells. Our optimized detergent-free buffer consisting of ammonium bicarbonate yielded comparable findings. For the same number of cells, 5000 proteins were identified from 29,000 peptides, 40% of which demonstrated a CV under 25%.