Decoupling Quantification and Identification for Label-Free Top-Down Proteomics Improves Proteome Coverage

Decoupling quantification and identification in label-free top-down proteomics more than doubled reliably quantified mass features and increased identification coverage by 50% via MS1-only quantification plus targeted MS2.

• Why it matters: Conventional TDP-LFQ is limited because quantification and identification compete for acquisition time, leaving missing values, insufficient fragmentation, and undersampling of low-abundance proteoforms.
• What they did: The workflow performs MS1-only acquisition for quantification, then applies targeted data-dependent MS2 identification to significantly differentially abundant features, optimizing MS2 parameters.
• The result: Across chromatographic conditions, MS setups, and sample preparation strategies, it improved quantitative precision and proteoform coverage, and in Escherichia coli grown on glucose or acetate enabled insights not accessible from a previously published bottom-up dataset.

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