Development of newer mass spectrometers are promoted to be faster and more sensitive than previous generations. Here we compare a wide range of instruments by DIA to benchmark their performance and optimize DIA methods across nano flow, cap flow and analytical flow to reduce acquisition time and maximise coverage.
HeLa digest (400 ng, n=3) was acquired using a standardized DIA nano flow method (66 min linear gradient, 15K MS2 resolution, 34 variable width windows) across 7 orbitrap instruments to compare the number of identifications returned. DIA method optimization was performed by varying MS2 resolution and the number of DIA windows to maximise identifications while still returning appropriate cycle times. Additionally, capillary flow DIA methods (1000 ng loading) were evaluated to determine if acquisition times could be reduced without compromising coverage. Finally, capillary flow and analytical flow methods on a 7600+ QTOF instrument were assessed for their speed and sensitivity.
The MS2 resolution and the number of DIA windows needs to be balanced against the cycle time in order to obtain adequate points under the peak for accurate quantitation. A higher MS2 resolution returns a higher number of identifications with better reproducibility. Capillary flow methods with higher loadings compared to nano flow return similar number of identifications with a shorter run time allowing for faster acquisition. The 7600+ QTOF gave the fastest acquisition time but poorest coverage compared to orbitrap instruments, but a 50 ng load by capillary flow (Evosep One, 1.5 µl/min, 11.5 min) gave comparable identifications to a 2000 ng load by analytical flow (LC40, 0.8 ml/min, 8 min).
When running studies, users must balance between run time, sensitivity, coverage, reproducibility and sample availability. Here we assessed a wide range of instruments and DIA methods to allow users to determine which combination of parameters would suit their experimental aims.