While great advances have been achieved in the performance of mass spectrometers, deep proteome coverage is still impaired for highly complex samples with high dynamic concentration ranges. To address this challenge a multitude of offline fractionation techniques are employed. However, these are time-consuming and mostly use higher sample amounts. Employing gas-phase fractionation with a FAIMS Pro interface on the new Orbitrap Exploris 480 mass spectrometer mitigates these challenges.
The data demonstrate that the singly charged ion population can be filtered out effectively by applying compensation voltages larger than -40 V. As a consequence, the chemical background can be significantly reduced even in single CV runs. This aspect is especially beneficial for the peptide species eluting at higher organic concentrations because of co-eluting contaminants.
Internal switching between CVs enhances the range of identifiable peptide ion species. With CV switching between -45 V, -55 V, and -65 V, we were able to boost protein identification by as much as 15 % in 90 min LC-gradients and 1 µg HeLa digest accompanied by 30 % more unique peptide IDs. With lower sample loads, we still gain 10% protein IDs even though peptide IDs are slightly decreased which points to the enhanced sampling with FAIMS. In fact, by internal switching of compensation voltages, the number of protein identifications can be increased in a single shot proteomic experiment comparable to a traditional 2D-LC setup.