Modular automated bottom-up proteomic sample preparation for high-throughput applications.

Area of Science:

• Microbiology
• Biochemistry
• Proteomics

Background:

• Manual proteomic sample preparation is time-consuming and can compromise data quality, especially for large-scale studies.
• Automated liquid handling systems offer a solution to improve throughput and reproducibility in sample preparation.

Purpose of the Study:

• To develop and validate a modular, automated protocol for bottom-up proteomic sample preparation of Gram-negative bacteria and fungal cells.
• To enable rapid, low-variance preparation of hundreds of samples using automated liquid handlers.

Main Methods:

• A three-part modular protocol was optimized for cell lysis, protein quantification, and normalization/tryptic digestion.
• The protocol was implemented on Beckman-Coulter Biomek automated liquid handlers.
• Validation involved analyzing 47 samples from Pseudomonas putida and Rhodosporidium toruloides.

Main Results:

• The automated workflow prepared 50 micrograms of protein from 96 samples for tryptic digestion in under an hour.
• Achieved protein relative quantity variance (CV%) below 15% for 94 peptide samples.
• Eliminated the need for desalting column cleanup in the sample preparation process.

Conclusions:

• The developed modular workflow provides robust and reproducible proteomic samples for high-throughput applications.
• This automated method significantly improves sample preparation efficiency for bacterial and fungal proteomics.
• The protocol is flexible and adaptable for future advancements in proteomic analysis.