An Integrated Experimental and Informatics Strategy Enables Large Proteoform Identification in Top-Down Proteomics

An integrated experimental and informatics workflow for human heart tissue identifies 703 nonredundant proteoforms from 236 proteins, including 22 proteoforms >50 kDa and extending to 223 kDa.

• Why it matters: Large proteoforms are hard to detect and interpret because signal-to-noise drops with molecular weight, coeluting smaller species interfere, precursor isotopic envelopes are unresolved, and existing informatics tools limit MS/MS interpretation.
• What they did: The approach combines online two-dimensional LC (size-exclusion plus reversed-phase), precursor mass correction for DDA with isotopically unresolved precursors, and large-protein-optimized DIA using intact-mass-guided candidate discovery and MS/MS validation.
• The result: By bridging detection-to-identification for large proteins, it expands the accessible mass range of discovery top-down proteomics and enables identification of proteoforms up to 223 kDa in human heart tissue.

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