Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes

Mass spectrometry-based quantitative
proteomic analysis has proven
valuable for clinical and biotechnology-related research and development.
Improvements in sensitivity, resolution, and robustness of mass analyzers have also
added value. However, manual sample preparation protocols are often
a bottleneck for sample throughput and can lead to poor reproducibility,
especially for applications where thousands of samples per month must
be analyzed. To alleviate these issues, we developed a “cells-to-peptides”
automated workflow for Gram-negative bacteria and fungi that includes
cell lysis, protein precipitation, resuspension, quantification, normalization,
and tryptic digestion. The workflow takes 2 h to process 96 samples
from cell pellets to the initiation of the tryptic digestion step
and can process 384 samples in parallel. We measured the efficiency
of protein extraction from various amounts of cell biomass and optimized
the process for standard liquid chromatography–mass spectrometry
systems. The automated workflow was tested by preparing 96 Escherichia coli samples and quantifying over 600
peptides that resulted in a median coefficient of variation of 15.8%.
Similar technical variance was observed for three other organisms
as measured by highly multiplexed LC-MRM–MS acquisition methods.
These results show that this automated sample preparation workflow
provides robust, reproducible proteomic samples for high-throughput
applications.