Lyophilization prior to homogenisation and extraction increases protein yield and membrane protein detection in Gram-negative bacterial proteomic analyses

Multi-drug resistant Gram-negative bacteria (GNB) are major contributors to the anti-microbial resistance (AMR) burden in humans and animals. AMR mechanisms are primarily mediated by proteoforms and, therefore, proteomic analyses of GNB offers a significant advantage in understanding the mechanisms of AMR. A large portion of these mechanisms are mediated by membrane proteins, however, they are often difficult to extract due to their hydrophobic nature and complex interactions with other components of the cell membrane. To extract the greatest number of proteoforms, an efficient homogenisation protocol is required to effectively disrupt the rigid cell wall and membrane. Bead-beating has previously been demonstrated to increase cellular disruption and improve protein extraction yields in mammalian systems, however, the efficiency of bead-beating is dependent on various factors, including cell structure characteristics. Using Escherichia coli and Klebsiella pneumoniae, we systematically compared the extraction efficiency of bead-beating with flash frozen and lyophilized cell pellets. We demonstrate that lyophilization prior to homogenisation by bead-beating increases protein extraction yield, and increases the detection of hydrophobic, membrane proteins. We detected numerous unique membrane proteins in each bacterial isolate, including ABC transporters, multi-drug efflux pumps, and proteins involved in lipopolysaccharide (LPS) synthesis, when lyophilizing prior to bead-beating compared to only flash freezing prior to bead-beating. As membrane proteins play a central role in AMR resistance mechanisms, this improvement in their isolation and identification will aid in under-standing the resistance and molecular mechanisms associated with multi-drug resistant GNB.