Multi-reflecting TOF MS for analyzing proteins

This paper presents detailed results of previously reported protein studies conducted using a prototype multi-reflecting time-of-flight mass spectrometer with orthogonal acceleration (o-MRT). The o-MRT instrument features a relatively short 5-m effective flight path to minimize gas scattering of protein ions. Despite this, the instrument achieves an impressive mass resolving power exceeding 200,000, fully resolving isotopic envelopes for proteins up to 80 kDa. For multiply charged ions of mid-sized proteins (up to 30 kDa), the instrument's resolution surpasses 250,000, enabling efficient charge and isotope deconvolution. This process consolidates multiple peaks from a specific proteoform into a single molecular weight peak, significantly simplifying protein spectra and facilitating accurate mass measurements. Mass analysis of horse myoglobin demonstrates exceptional mass precision, achieving 100 ppb accuracy for the monoisotopic mass. The o-MRT instrument exhibits a sensitivity of 10⁴ ions/fmol, allowing for the detection of horse myoglobin from 100 pM to 10 μM concentration range. It accurately identifies known proteoforms in exemplar mixtures of seven proteins without chromatography, demonstrating a dynamic range of 1000. The o-MRT instrument showcases versatility in MS/MS analysis, achieving over 80 % sequence coverage for proteoforms. In MS/MS analyses of horse myoglobin and human hemoglobin chain A, mass accuracy of 0.5 ppm is maintained over a dynamic range of 1000. With future coupling to liquid chromatography, the compact o-MRT instrument is expected to be a valuable tool for identifying proteins within moderately complex mixtures.