Proteasome is a large protein complex responsible for protein degradation in mammalian cells. It is important to determine the protein complex structure for better understanding of its catalytic function. There are multiple analytical methods could be used to access the protein structure . In recent years HDX-MS has emerged as a powerful tool for studying protein, protein complex’s conformation and conformation dynamics. In this presentation, HDX-MS was used to study the structure conformation of 20S proteasome complex in presence/absence of SDS.
Rabbit 20S proteasome protein complex and protein complex with 0.1% SDS samples were labeled with deuteration buffer and incubated for multiple time points. The deuterated samples were then quenched and digested online with a pepsin column in a fully automated manner. The digested peptides were injected into a reverse phase column with a short gradient. MS analysis was performed with Thermo ScientificTM Orbitrap LumosTM mass spectrometer. Peptides mapping and PTM analysis were performed with Thermo Scientific™ BioPharma Finder 3.1. HDX experimental data were analyzed by BioPharma Finder 3.1, Mass Spec Studio (Dr. Schriemer, University of Calgary).
The 20S proteasome complex contains 28 subunits arranged into four stacked rings: seven alpha non-catalytic subunits and seven beta-subunits . All seven alpha and seven beta-subunits were identified with sequence coverage range from around 79% to 93%. The identified peptides were used to calculate the deuterate incorporation of the proteasome with/without SDS in two protein states. The deuterium incorporation varied indicating the proteins conformed with different solvent associability. Overall, the presence of SDS caused most of the protein regions to have more of deuterium incorporation. The protection factors were calculated by BioPharma Finder 3.1 which reviewed the protein’s solvent accessibility on amino acid level. Structural information obtained by HDX-MS for 20S proteasome complex dynamics was validated using structure obtained through electron microscopy.