Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition

Proteomics has become an increasingly important tool
in medical
and medicinal applications. It is necessary to improve the analytical
throughput for these applications, particularly in large-scale drug
screening to enable measurement of a large number of samples. In this
study, we aimed to establish an ultrafast proteomic method based on
5-min gradient LC and quadrupole-Orbitrap mass spectrometer (Q-Orbitrap
MS). We precisely optimized data-independent acquisition (DIA) parameters
for 5-min gradient LC and reached a depth of >5000 and 4200 proteins
from 1000 and 31.25 ng of HEK293T cell digest in a single-shot run,
respectively. The throughput of our method enabled the measurement
of approximately 80 samples/day, including sample loading, column
equilibration, and wash running time. We demonstrated that our method
is applicable for the screening of chemical responsivity via a cell
stimulation assay. These data show that our method enables the capture
of biological alterations in proteomic profiles with high sensitivity,
suggesting the possibility of large-scale screening of chemical responsivity.