Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial...

Area of Science:

• Proteomics
• Microbiology
• Biochemistry

Background:

• Proteome analysis is frequently limited by the availability of small bacterial cell numbers, especially from in vivo or environmental samples.
• Existing sample preparation protocols may not be optimal for low-input bacterial samples, hindering comprehensive proteomic studies.

Purpose of the Study:

• To adapt and optimize the single pot solid-phase enhanced sample preparation (SP3) protocol for enhanced proteome analysis of bacteria in limited quantities.
• To improve peptide and protein identification rates and ensure reliable quantification for low-biomass bacterial samples.

Main Methods:

• Adaptation of the single pot solid-phase enhanced sample preparation (SP3) protocol.
• Integration of effective cell disruption using detergents for sample preparation.
• Comparative analysis against established strain-specific protocols using Staphylococcus aureus, Streptococcus suis, and Legionella pneumophila.

Main Results:

• The adapted SP3 protocol significantly increased peptide and protein identifications across various bacterial species.
• A substantial increase of up to 300% in protein and 620% in peptide identifications was observed for Legionella pneumophila.
• Improved reproducibility in protein quantification and enhanced data completeness between replicates were achieved.

Conclusions:

• The optimized SP3 protocol effectively overcomes limitations associated with low bacterial cell numbers in proteomic analyses.
• This method enables comprehensive proteomics for diverse bacterial species, including Gram-positive and Gram-negative types.
• The protocol is valuable for analyzing low bacterial cell numbers from various settings, such as infection assays and environmental samples.