PulseDIA: Data-Independent Acquisition Mass Spectrometry Using Multi-Injection Pulsed Gas-Phase Fractionation

Area of Science:

• Proteomics
• Mass Spectrometry
• Analytical Chemistry

Background:

• Data-independent acquisition (DIA) mass spectrometry (MS) performance relies on peptide precursor separation efficiency.
• Orbitrap-based MS has limitations in separation efficiency due to slower scanning rates compared to time-of-flight (TOF)-based MS.

Purpose of the Study:

• To introduce PulseDIA, a multi-injection gas-phase fractionation (GPF) strategy to enhance DIA-MS.
• To improve peptide and protein identification sensitivity and reproducibility in proteomic analyses.

Main Methods:

• PulseDIA divides conventional DIA analysis into multiple injections with complementary windows.
• The method was tested using mouse liver digests and human cholangiocarcinoma (CCA) tissue samples.

Main Results:

• PulseDIA identified up to 50% more peptides and 29% more protein groups than conventional DIA in mouse liver digests.
• Compared to conventional multi-injection GPF, PulseDIA identified more peptides and protein groups with higher flexibility.
• In CCA samples, PulseDIA identified 7796 protein groups, a 14% increase over conventional DIA, with reduced missing values and detection of novel dysregulated proteins.

Conclusions:

• PulseDIA is an effective strategy for enhancing DIA-MS sensitivity and reproducibility.
• The method offers significant improvements in peptide and protein identification, particularly for complex proteomic samples.
• PulseDIA advances proteomic profiling in applications like cancer research, revealing previously undetected biomarkers.