A Simplified Label-Free Method for Proteotyping Sets of Six Isolates in a Single Liquid Chromatography-High-Resolution Tandem Mass Spectrometry Analysis

Clinical
diagnostics and microbiology require high-throughput identification
of microorganisms. Sample multiplexing prior to detection is an attractive
means to reduce analysis costs and time-to-result. Recent studies
have demonstrated the discriminative power of tandem mass spectrometry-based
proteotyping. This technology can rapidly identify the most likely
taxonomical position of any microorganism, even uncharacterized organisms.
Here, we present a simplified label-free multiplexing method to proteotype
isolates by tandem mass spectrometry that can identify six microorganisms
in a single 20 min analytical run. The strategy involves the production
of peptide fractions with distinct hydrophobicity profiles using spin
column fractionation. Assemblages of different fractions can then
be analyzed using mass spectrometry. Results are subsequently interpreted
based on the hydrophobic characteristics of the peptides detected,
which make it possible to link each taxon identified to the initial
sample. The methodology was tested on 32 distinct sets of six organisms
including several worst-scenario assemblages–with differences
in sample quantities or the presence of the same organisms in multiple
fractions–and proved to be robust. These results pave the way
for the deployment of tandem mass spectrometry-based proteotyping
in microbiology laboratories.