Improving Proteomic Identification Using Narrow Isolation Windows with Zeno SWATH Data-Independent Acquisition

Area of Science:

• Proteomics and Mass Spectrometry
• Biomolecular Analysis
• Data-Independent Acquisition (DIA)

Background:

• Sequential window acquisition of all theoretical mass spectra (SWATH) is a preferred data-independent acquisition (DIA) technique for proteomic analysis.
• Optimizing SWATH parameters is crucial for enhancing proteomic identification depth and data quality.

Purpose of the Study:

• To optimize the SWATH-DIA method using a narrow isolation window placement approach, termed narrow SWATH (nSWATH).
• To evaluate the performance of nSWATH in terms of protein identification and quantification accuracy in complex biological samples and benchmark mixtures.

Main Methods:

• Optimization of SWATH-DIA acquisition parameters, specifically narrow isolation windows (1.9 and 2.9 Da) on a ZenoTOF 7600 mass spectrometer.
• Analysis of acquired data using DIA-NN software (version 1.8.1).
• Testing nSWATH performance on digested peptides from mouse liver and HEK293T cells, as well as benchmark samples containing mixtures of *Escherichia coli*, yeast, and human peptides.

Main Results:

• nSWATH identified significantly more protein groups compared to conventional SWATH: 7.52% improvement for mouse liver and 4.99% for HEK293T cells (400 ng protein).
• nSWATH achieved high accuracy and precision in benchmark samples, identifying an average of 9342 protein groups (12.6% improvement) and 95,456 precursors (9.63% improvement) at 200 ng.
• The method demonstrated improved identification across various loading amounts, with a 40.7% increase in protein groups identified at 25 ng.

Conclusions:

• The optimized nSWATH method significantly enhances proteomic identification depth and data quality in complex samples.
• nSWATH offers comparable accuracy and precision to conventional SWATH while improving protein group identification rates.
• This optimized DIA strategy contributes to deeper proteomic profiling of biological samples.