Toward Comprehensive Plasma Proteomics by Orthogonal Protease Digestion.

Area of Science:

• Clinical proteomics and mass spectrometry
• Biomarker discovery for infectious diseases

Background:

• Accurate protein identification in large clinical studies is crucial for proteomics.
• Data-independent acquisition (DIA/SWATH-MS) offers reproducibility but relies on spectral libraries.
• Existing methods for spectral library generation can be limiting for comprehensive proteome analysis.

Purpose of the Study:

• To establish an efficient workflow for generating spectral libraries for plasma proteome analysis.
• To comprehensively profile the plasma proteome in individuals with and without active tuberculosis (TB).
• To identify novel diagnostic biomarkers for TB, including intracellular and previously undetectable proteins.

Main Methods:

• A tip-based workflow was employed for spectral library generation.
• Utilized a combination of proteases (trypsin, GluC, AspN) to enhance peptide coverage.
• Applied the generated library to quantify plasma proteome differences in TB patients using DIA/SWATH-MS.

Main Results:

• Identified over 30,000 peptides corresponding to 3309 proteins.
• Quantified over 400 differentially expressed proteins in TB patients within 50 minutes of MS acquisition.
• Detected diagnostic *Mycobacterium tuberculosis* (Mtb) proteins and novel intracellular plasma proteins.

Conclusions:

• The developed spectral library workflow significantly improves plasma proteome coverage and protein identification.
• This method enables rapid quantification of plasma proteome differences, facilitating TB biomarker discovery.
• The findings highlight the potential of this approach for identifying novel diagnostic markers for TB.