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

The performance of data-independent acquisition (DIA) mass spectrometry (MS) depends on the separation efficiency of peptide precursors. In Orbitrap-based mass spectrometers, separation efficiency of peptide precursors is limited by the relatively slow scanning rate compared to time of flight (TOF)-based MS. Here, we present PulseDIA, a multi-injection gas-phase fractionation (GPF) strategy for enhanced DIA-MS. This is achieved by equally dividing the conventional DIA analysis covering the entire mass range into multiple injections for DIA analyses with complementary windows. Using mouse liver digests, the PulseDIA method identified up to 50% more peptides and 29% more protein groups than that by conventional DIA with the same length of effective gradient time. Compared to conventional multi-injection GPF, PusleDIA exhibited higher flexibility and identified up to 18% more peptides and 8% more protein groups using two injections. The gain of peptides per effective time unit was the highest in PulseDIA compared to conventional DIA and GPF. We further applied the PulseDIA method to profile the proteome of 18 human tissue samples (benign and malignant) from nine cholangiocarcinoma (CCA) patients. PulseDIA identified 7796 protein groups in these CCA samples, with a 14% increase of protein group identification compared to the conventional DIA method. The missing value for protein matrix dropped by 7% using PulseDIA compared to DIA. A total of 681 significantly altered proteins were detected in CCA samples using PulseDIA, including several dysregulated proteins, which were absent in the conventional DIA analysis. Taken together, we present PulseDIA as an enhanced DIA-MS method with improved sensitivity and reproducibility.