Multiplexing the Identification of Microorganisms via Tandem Mass Tag Labeling Augmented by Interference Removal...

Area of Science:

• Microbiology
• Proteomics
• Analytical Chemistry

Background:

• Accurate microorganism identification is crucial for public health, research, and safety.
• Mass spectrometry-based proteomics offers effective microbial identification but is limited by long analysis times (approx. 1 hour/sample).
• This time constraint hinders routine clinical diagnostics and culturomics.

Purpose of the Study:

• To develop a faster method for microorganism identification using mass spectrometry.
• To address the time limitations of current proteomic identification techniques.
• To improve the efficiency of microbial analysis for clinical and research settings.

Main Methods:

• Employed tandem mass tags (TMTs) for multiplex identification of microorganisms in a single MS/MS experiment.
• Utilized a modified expectation maximization (EM) algorithm to correct for interference in TMT reporter ion intensities.
• Validated the method using 94 MS/MS experiments with 1931 true positive and 317 true negative TMT-labeled channels.

Main Results:

• Achieved high species-level identification sensitivity of 93-97% and 100% specificity.
• Demonstrated a significant reduction in mass spectrometer time per sample: to 10 min (TMT-6) and 6 min (TMT-10) from 1 hour.
• Showcased compatibility with advanced mass spectrometers like Orbitrap Astral for further time reduction.

Conclusions:

• The proposed TMT-based multiplexing method with EM correction dramatically accelerates microorganism identification.
• This advancement overcomes a major bottleneck in clinical diagnostics and culturomics.
• The method offers a faster, accurate, and broad-spectrum solution for microbial analysis.