High-throughput cell-based proteomics perturbation: HTP Perturbation

ABSTRACT: We have developed a high-throughput (HTP) cell-based workflow for the accurate quantification of proteins from approximately 100 samples per day. This bottom-up proteomics workflow automates the processing of cells grown in 96-well plates for liquid-chromatography mass spectrometry (LC-MS) analysis. The HTP system leverages the LE220-plus focused-ultrasonicator system (Covaris) for cell lysis and solubilization in tandem with the Biomek i7 automated liquid handling workstation (Beckman) for subsequent peptide processing prior to LC-MS. AC16 human cardiomyocytes were cultured in a 96-well plate under optimized conditions that was compatible with downstream HTP cell-based assay investigation. Sonication parameters and solubilization buffers were evaluated. An average protein concentration of 0.5 microg/microl was obtained from approximately 38,000 cells and optimal cell lysis was observed in 5 % SDS. SDS-based extraction resulted in more than 70,000 peptides to identify 4,300 proteins, with 75 % of proteins measured at an average coefficient of variation (CV) under 25%. Our HTP system was also adapted to detect proteins from low peptide loads or with short LC-MS acquisition gradients. Our HTP protocol was further evaluated in a cell model of cardiac ischemic reperfusion. Proteomic analysis of differentially expressed proteins (DEPs) demonstrated significant down-regulation in hypoxic state and an influx in reperfused condition, with most upregulated proteins involved in lipid and metabolic metabolism, the main drivers after hypoxic cellular injury. The developed HTP pipeline can be applied to both label free and stable isotope-labeling as well as investigating important key regulators in major cell-based assays, including post-translational modifications. Furthermore, drug targets and cellular perturbations could be rapidly investigated.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jennifer Van Eyk  

PROVIDER: MSV000095291 | MassIVE | Wed Jul 10 13:21:00 BST 2024

SECONDARY ACCESSION(S): PXD053859

REPOSITORIES: MassIVE