Toward Comprehensive Plasma Proteomics by Orthogonal Protease Digestion

Rapid and consistent protein identification
across large clinical
cohorts is an important goal for clinical proteomics. With the development
of data-independent technologies (DIA/SWATH-MS), it is now possible
to analyze hundreds of samples with great reproducibility and quantitative
accuracy. However, this technology benefits from empirically derived
spectral libraries that define the detectable set of peptides and
proteins. Here, we apply a simple and accessible tip-based workflow
for the generation of spectral libraries to provide a comprehensive
overview on the plasma proteome in individuals with and without active
tuberculosis (TB). To boost protein coverage, we utilized nonconventional
proteases such as GluC and AspN together with the gold standard trypsin,
identifying more than 30,000 peptides mapping to 3309 proteins. Application
of this library to quantify plasma proteome differences in TB infection
recovered more than 400 proteins in 50 min of MS acquisition, including
diagnostic Mycobacterium tuberculosis (Mtb) proteins that have previously been detectable primarily by
antibody-based assays and intracellular proteins not previously described
to be in plasma.