Multiplexed Single Ion Mass Spectrometry Improves Measurement of Proteoforms and Their Complexes Jared O. Kafader, † Rafael D. Melani, † Kenneth R. Durbin, † Bon Ikwuagwu, † Bryan P. Early, † Ryan T. Fellers, † Steven C. Beu, ⊥ Vlad Zabrouskov, ‡ Alexander A. Makarov, § Joshua T. Maze, # Deven L. Shinholt, # Ping F. Yip, ‡ Danielle Tullman-Ercek, † Michael W. Senko, ‡ Philip D. Compton, * , † Neil L. Kelleher * , † † Departments of Chemistry, Molecular Biosciences, and Chemical and Biological Engineering, the Chemistry of Life Processes Institute, the Proteomics Center of Excellence at Northwestern University, Evanston, Illinois 60208, United States ⊥ S.C. Beu Consulting, Austin, Texas 78729, United States ‡ Thermo Fisher Scientific, San Jose, California 95134, United States # Thermo Fisher Scientific, Austin, Texas 78728, United States § Thermo Fisher Scientific, Bremen 28199, Germany *Corresponding authors: philip-compton@northwestern.edu n-kelleher@northwestern.edu Abstract: A new Orbitrap-based single ion analysis procedure is shown to be possible by determining the direct charge on numerous measurements of individual protein ions to generate true mass spectra. The deployment of an Orbitrap system for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by traditional measurement of an ensemble of ions. . CC-BY-NC-ND 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted July 26, 2019. . https://doi.org/10.1101/715425 doi: bioRxiv preprint