Single cell proteomics (SCP) has advanced significantly for eukaryotic cells, enabling the identification of thousands of proteins per cell. However, bacteria present a far greater technical challenge: they are roughly 1,000 times smaller than eukaryotic cells and contain well under 1 pg of protein per cell, making existing SCP workflows insufficiently sensitive. Until now, no study had demonstrated detection of biologically meaningful protein regulation at the level of a single bacterium.

Leodolter et al. aimed to establish a proof-of-concept bacterial SCP (bacSCP) workflow capable of detecting protein-level responses to stress in individual bacterial cells. To address this, the researchers adapted a label-free, lossless “One-Pot” sample preparation workflow, including fluorescence-guided cell sorting via the cellenONE robot, combined with a highly sensitive LC-MS platform performing global, label-free quantitative proteomics.

As early adopters of the prototype NanoShield® 5×100 NZ C18 trap column, the researchers paired it with an Aurora® Rapid™ 8×75 XT C18 UHPLC column, on a Vanquish Neo with an EASY-Spray source and an Orbitrap Astral with a FAIMS Pro Duo interface. The NanoShield® trap column and Aurora® Rapid™ 8×75 XT column underpin the LC configuration used in this bacSCP workflow, enabling proteome measurements at unprecedented depth from single bacterial cells. The authors conclude the workflow is sufficiently sensitive and robust to detect heat shock response at the single-bacterium level, demonstrating performance in one of the most challenging low-input regimes for LC-MS proteomics.

Leveraging this workflow, the researchers demonstrated that the heat shock chaperones GroEL, GroES, and ClpC are significantly upregulated in heat-stressed Bacillus subtilis at the single-cell level, with a subset of four cells displaying a markedly stronger stress response, providing the first evidence of proteomic heterogeneity in individual bacteria under stress.

This work opens a new frontier for studying antibiotic tolerance, persister cell formation, and bacterial pathogenicity at the single-cell proteome level.

Publication
bioRxiv

Authors

Julia Leodolter, Tim Thierer, Karl Mechtler, & Manuel Matzinger;

Title

Pushing the limits of SCP: bacSCP, a proof-of-concept study to investigate heterogeneity of bacteria by single cell proteomics