Identifying Subcellular Structure Components in Escherichia Coli by Crosslinking and SEC-MS

Area of Science:

• Cellular biology
• Proteomics
• Biochemistry

Background:

• Cells contain diverse structures crucial for biochemical and signaling processes.
• Current techniques for quantifying or discovering subcellular structures are limited.
• Understanding cellular organization requires advanced methods to identify molecular assemblies.

Purpose of the Study:

• To explore a proteomics approach combining chemical crosslinking, size-exclusion chromatography, and mass spectrometry (SEC-MS) for discovering subcellular structures.
• To apply formaldehyde crosslinking-assisted SEC-MS to a bacterial system for the first time.
• To identify and characterize protein assemblies and subcellular bodies in bacteria.

Main Methods:

• Utilized formaldehyde crosslinking to preserve weak molecular interactions within whole cell lysates.
• Employed size-exclusion chromatography coupled with mass spectrometry (SEC-MS) to separate and analyze cellular components.
• Applied the method to E. coli, including ectopic expression of the FUS assembly protein.

Main Results:

• Demonstrated detection of large FUS protein structures in E. coli using SEC-MS.
• Showed formaldehyde crosslinking enriches E. coli proteins in large and medium-sized particles.
• Identified known E. coli protein assemblies and condensates, alongside potentially novel prokaryotic metabolic associations with subcellular bodies.

Conclusions:

• Formaldehyde crosslinking-assisted SEC-MS is a viable, unbiased method for analyzing bacterial cellular structures.
• This technique can identify known and potentially novel subcellular assemblies and bodies.
• The method holds promise for supplementing targeted approaches in discovering new cellular structures across various cell types.