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

ABSTRACT: 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.

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ORGANISM(S): Escherichia Coli

SUBMITTER: Breyer-Lynne Woodland

LAB HEAD: Matthew P. Padula

PROVIDER: PXD075198 | Pride | 2026-03-08

REPOSITORIES: Pride

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Project description:The identification of putative biomarkers for predicting abdominal aortic aneurysm (AAA) progression can be achieved through proteomic strategies, particularly with shotgun proteomics. However, the heterogeneity of AAA tissue poses challenges for tissue homogenization and protein extraction, compromising reproducibility, an essential factor for clinical translation. Thus, we aimed to optimize a protocol for AAA tissue homogenization and protein extraction and to develop a standard operating procedure (SOP) to maximize protein yield and foster reproducibility in the field. In this context, several experimental variables were systematically tested in a bead-beating method for tissue homogenisation, including bead size (1.4 mm versus 2.8 mm of diameter), the number of extraction cycles (one up to three), a bead-to-tissue mass ratio at 20, 30, or 40, the lysis buffer-to-tissue ratio (10, 15, or 20 μL/mg), and the lysis buffer composition (RIPA, Urea/thiourea, and HEPES). Optimal conditions for protein extraction were achieved using 1.4 mm zirconium dioxide beads in two homogenization cycles, with a bead-to-tissue mass ratio of 30:1 and a lysis buffer-to-tissue ratio of 20 μL lysis buffer per milligram of tissue, in 2 mL O-ring cryotubes. Depending on the purpose of downstream analysis, recommendations for the use of each lysis buffers were provided based on their specific performance across specific indexes. In this context, HEPES and Urea/Thiourea buffers are recommended for studies prioritizing quantification reproducibility, while RIPA buffer is advised for protein extraction reproducibility. As a proof of concept, the optimized SOP was applied to characterize the AAA tissue proteome and several processes and pathways relevant in AAA pathophysiology were highlighted, including blood coagulation, cellular responses to stress, neutrophil degranulation, wound healing, and pathways of the immune system. Additionally, the enrichment analysis identified ECM remodeling and platelet activation, and degranulation as strongly represented pathways across the functional analysis for each buffer, reinforcing their contribution to aneurysm pathophysiology. Hence, these findings demonstrated that the SOP is well-suited for identifying disease-relevant biomarkers and therapeutic targets.

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

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