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.