Dynamic Quadrupole Selection to Associate Precursor Masses with MS/MS Products in Data-Independent Acquisition

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Data-independent acquisition (DIA) mass spectrometry enables high-throughput proteomic analyses.
• Conventional DIA methods often co-select multiple precursor ions, leading to chimeric spectra and reduced accuracy.
• Resolving co-isolated precursors is crucial for improving the reliability of bottom-up proteomic studies.

Purpose of the Study:

• To develop and evaluate a novel DIA mass spectrometry method for improved precursor ion identification.
• To enhance the resolution of co-isolated precursor ions in complex proteomic samples.
• To assess the method's performance using various calibrants and sample types.

Main Methods:

• A method varying quadrupole selection width during ion accumulation was implemented.
• Scan-to-scan product ion intensity profiles were used to infer precursor mass by overlapping selection windows.
• The technique was tested using internal calibrants and a tryptic-digest monoclonal antibody sample on Q-Orbitrap mass analyzers.
• Direct infusion and liquid chromatography were employed for sample analysis.

Main Results:

• The method successfully coupled product ion intensity to precursor ion mass.
• Overlapping selection windows enabled the inference of precursor mass from product ion profiles.
• With direct infusion, precursors separated by 1 Th were resolved using 10 Th windows with 5 Th overlap.
• Product ions were associated within 0.3 Th of their precursor m/z, yielding a precursor ion m/z resolving power of ~33.

Conclusions:

• The described method effectively resolves co-isolated precursor ions in DIA mass spectrometry.
• This technique significantly improves the precursor ion m/z resolving power, enhancing proteomic data accuracy.
• The method demonstrates broad applicability for analyzing complex proteomic samples, including antibody digests.