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

Area of Science:

• Mass spectrometry
• Proteomics
• Analytical chemistry

Background:

• Data-independent acquisition (DIA) proteomics primarily uses beam-type collision-induced dissociation (HCD) due to its speed.
• HCD's reliance on charge state calibration limits its effectiveness in mixed-charge DIA isolation windows.
• Resonance-excitation collision-induced dissociation (reCID) offers charge-independent activation but was historically too slow for DIA.

Purpose of the Study:

• To evaluate resonance-excitation collision-induced dissociation (reCID) as a practical fragmentation method for data-independent acquisition (DIA) proteomics.
• To assess if modified mass spectrometry instrumentation can enable reCID acquisition rates comparable to HCD.
• To compare the performance of reCID and HCD in terms of precursor and protein detection, and quantitative accuracy in DIA proteomics.

Main Methods:

• Utilized a modified Orbitrap Tribrid Apex MultiOmics mass spectrometer (Apex) to achieve HCD-comparable acquisition rates with reCID.
• Performed matched acquisition rate experiments on tryptic HeLa digests using both reCID and HCD.
• Employed Carafe fine-tuned, fragmentation-matched spectral libraries for data analysis.
• Assessed peptide-level quantitative performance and protein abundance rankings across multiple cancer cell lines and pooled mixtures.

Main Results:

• The modified Apex instrument enabled reCID acquisition rates comparable to HCD.
• reCID achieved precursor and protein detection levels similar to HCD when using fine-tuned spectral libraries.
• Library fine-tuning yielded greater improvements in reCID precursor detections (24%) compared to HCD (5%).
• Peptide-level quantitative performance, including ions per peptide, precision, and accuracy, was maintained with reCID.
• Protein abundance rankings were highly conserved between Apex reCID and HCD methods, and across different mass spectrometry platforms.

Conclusions:

• Resonance-excitation collision-induced dissociation (reCID) is a practical and effective fragmentation mode for DIA proteomics.
• Optimized instrumentation and spectral library tuning can overcome historical limitations of reCID speed and performance.
• reCID provides a viable alternative to HCD for DIA, offering comparable or improved performance in certain aspects.