A Robust Strategy for High-Throughput and Deep Proteomics by Combining Narrow-Window Data-Independent Acquisition and...

Area of Science:

• Proteomics
• Mass Spectrometry
• Analytical Chemistry

Background:

• Data-independent acquisition (DIA) mass spectrometry improves proteome coverage but struggles with isobaric labeling due to coisolation interference.
• Accurate quantification of reporter ions is crucial for multiplexed proteomics but is degraded by interference in standard DIA methods.

Purpose of the Study:

• To develop an ultra-narrow-window DIA workflow compatible with 18-plex TMTpro labeling for improved proteomic analysis.
• To overcome limitations in conventional isobaric labeling-based DIA pipelines for enhanced quantitative precision and accuracy.

Main Methods:

• Utilized an Orbitrap Astral mass spectrometer with ultra-narrow isolation windows (0.6 Th) and high MS/MS scan speed (200 Hz).
• Implemented mPE-MMR for accurate precursor mass assignment to multiplexed DIA spectra.
• Applied the workflow to ovarian cancer tissue digests for peptide and protein identification and quantification.

Main Results:

• Achieved DDA-level precursor specificity with ultra-narrow DIA windows.
• Identified substantially more peptides and protein groups compared to data-dependent acquisition (DDA).
• Sustained high reporter ion precision and accuracy, demonstrating robust quantitative performance.

Conclusions:

• The novel ultra-narrow-window DIA method significantly enhances proteomic depth and quantitative accuracy for isobaric-labeled samples.
• This approach overcomes major limitations in conventional DIA, offering deeper proteomic coverage without compromising quantitative robustness.
• The method shows great potential for large-scale clinical and population studies in proteomics.