Nested data independent MS/MS acquisition.

Area of Science:

• Proteomics
• Analytical Chemistry
• Mass Spectrometry

Background:

• Data independent acquisition (DIA) aims for comprehensive MS/MS data within chromatographic peak elution times.
• Current mass spectrometry (MS) technology faces limitations in achieving these DIA goals.
• Existing DIA methods like SWATH can produce chimeric spectra, complicating data interpretation.

Purpose of the Study:

• To propose and evaluate a new multiplex-based approach for enhanced DIA.
• To reduce chimeric product ion spectra in DIA experiments.
• To improve the comprehensiveness of MS/MS data acquisition in proteomics.

Main Methods:

• A customized Q Orbitrap instrument was programmed for a novel multiplex-based DIA technique.
• Multiple nonadjacent mass range segments were sequentially collected and fragmented.
• A nested design systematically varied mass range segments for mathematical assignment of product ions.
• The proposed method uses narrower mass windows compared to conventional DIA (SWATH).

Main Results:

• The nested DIA technique requires only one additional multiplexed scan to halve the mass window width, unlike conventional DIA.
• For a given cycle time, the proposed nested DIA produced significantly fewer chimeric product ion spectra than conventional DIA.
• The mathematical assignment of product ions within a narrow precursor mass range was enabled by the nested design.

Conclusions:

• The proposed nested DIA approach offers a significant reduction in chimeric spectra compared to conventional DIA.
• Further advancements in programming and hardware are necessary for fully comprehensive MS/MS data.
• This method represents a step towards achieving the goals of high-quality, comprehensive DIA in mass spectrometry.