Multi-Enzyme Data-Independent Acquisition Mass Spectrometry for Proteoform-Aware Single-shot Proteomics

mE-DIA multiplexes multi-enzyme proteoform-resolved DIA in single-shot injections, increasing identified protein groups by 30% and achieving 47% more protein groups quantified at ≤20% CV on shared data.

• Why it matters: Conventional bottom-up DIA discovery often misses sequence-level variation from splicing, post-translational modification, and proteolysis because multi-enzyme approaches require multiple LC-MS runs, library building, and manual cross-tool reconciliation.
• What they did: The mE-DIA workflow multiplexes complementary protease digests into one DIA injection and uses a library-free pipeline in a single analysis engine, with proteoform inference plus an interactive Peptide Mapper viewer.
• The result: In Streptococcus pyogenes, a three-enzyme multiplex resolved 990 protein-backbone variants among 309 canonical entries, 1.8-fold more than the best single-enzyme analysis, including an H69 C-terminal uS19 proteoform not resolved by trypsin-only.

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