Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mass Spectrometer

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange dataset PXD001305). We find the ultra-high-field Orbitrap mass analyzer capable of attaining a sequencing speed above 20 Hz and it routinely exceeds 10 peptide spectrum matches per second or up to 600 new peptides sequenced per gradient minute. We identify 4400 proteins from one microgram of HeLa digest using a one hour gradient, which is an approximately 30% improvement compared to previous instrumentation. In addition, we show very deep proteome coverage can be achieved in less than 24 hours of analysis time by offline high pH reversed-phase peptide fractionation from which we identify more than 140,000 unique peptide sequences. This is comparable to state-of-the-art multi-day, multi-enzyme efforts. Finally the acquisition methods are evaluated for single-shot phosphoproteomics, where we identify 7600 unique HeLa phospho-peptides in one gradient hour and find the quality of fragmentation spectra to be more important than quantity for accurate site assignment.