A Novel Ultrahigh-Resolution Y-Injection Multireflecting Time-of-Flight Mass Spectrometer for Bottom-Up Proteomics

The first results of using a new type of ultrahigh-resolution mass analyzer based on a planar multipass time-of-flight mass spectrometer with periodic reflecting lenses (Y-MRT MS) for bottom-up whole-proteome analysis are presented. The instrument achieves a resolving power in a range of 600,000-800,000 for peptide ions across the whole m/z range, with a high repetition rate of 300 Hz (averaged to 0.5-4 Hz for enhanced dynamic range). In preliminary experiments for human cell lines, MCF-7 and HeLa, single-shot 30 min gradient HPLC separations of 1 μg proteolytic digests yielded, on average, over 4000 protein groups in MS/MS-free proteome analyses using the DirectMS1 method. Combining three technical runs increased these numbers to 4500 protein groups at 1% FDR. Peptide ion mass measurements demonstrated an accuracy of 70-130 ppb across the whole m/z range, with a dynamic range exceeding 10⁴. In DIA mode (SWATH-DIA, 20 Th window, 30 min gradient), 4350 protein IDs were obtained at 1% FDR on average in single-shot LC-MS/MS runs. These results highlight the Y-MRT mass analyzer's potential for bottom-up proteomics. Further improvements in proteome coverage and analysis time are anticipated with optimized HPLC configurations and the integration of gas-phase ion mobility separation.