Revisiting resonance-excitation collision-induced dissociation for data-independent acquisition.

Data-independent acquisition (DIA) proteomics relies almost exclusively on beam-type collision-induced dissociation (HCD) because its short activation time supports fast acquisition rates required for DIA. However, HCD requires charge state calibration and is therefore imperfect for mixed-charge DIA isolation windows. Resonance-excitation collision-induced dissociation (reCID) offers a promising alternative to HCD for DIA because the ion activation is effectively independent of charge state. Historically, reCID's longer activation time has been considered too slow for DIA. Here, we revisit reCID for DIA proteomics using a modified Orbitrap Tribrid Apex MultiOmics mass spectrometer (Apex) that recovers acquisition-matrix overhead as additional ion injection time, enabling reCID acquisition rates comparable to HCD. Using matched acquisition rate settings of tryptic HeLa digests, reCID achieved precursor and protein detections similar to HCD when used with Carafe fine-tuned, fragmentation-matched spectral libraries. Library fine-tuning improved reCID precursor detections more than HCD detections, 24% versus 5%, indicating that HCD-trained prediction models are suboptimal for reCID spectra. ReCID also maintained peptide-level quantitative performance, including ions measured per peptide, precision, and accuracy. Across seven NCI cancer cell lines and pooled mixtures, protein abundance rankings were highly conserved between the Apex reCID and HCD methods, and also across Orbitrap Astral Zoom platforms. These results support reCID as a practical fragmentation mode for DIA proteomics.