An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method 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 typical measurements of ensembles of ions.
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Datasets utilized for the I2MS analyses can be found on the MassiVE repository, MSV000083840. Source data are available for Figs. 2 and 3 and Supplementary Figs. 2–4 and 9. Additional desired data that support the charge-determination findings of this study are available from the corresponding authors upon request.
Custom compiled code associated with the I2MS creation process is available via Supplementary Software or from the corresponding authors upon reasonable request.
Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).
Elliott, A. G., Harper, C. C., Lin, H.-W. & Williams, E. R. Mass, mobility and MSn measurements of single ions using charge detection mass spectrometry. Analyst 142, 2760–2769 (2017).
Keifer, D. Z., Pierson, E. E. & Jarrold, M. F. Charge detection mass spectrometry: weighing heavier things. Analyst 142, 1654–1671 (2017).
Benner, W. H. A gated electrostatic ion trap to repetitiously measure the charge and m/z of large electrospray ions. Anal. Chem. 69, 4162–4168 (1997).
Schmidt, H. T., Cederquist, H., Jensen, J. & Fardi, A. Conetrap: a compact electrostatic ion trap. Nucl. Instrum. Methods Phys. Res., Sect. B 173, 523–527 (2001).
Makarov, A. & Denisov, E. Dynamics of ions of intact proteins in the orbitrap mass analyzer. J. Am. Soc. Mass. Spectrom. 20, 1486–1495 (2009).
Rose, R. J., Damoc, E., Denisov, E., Makarov, A. & Heck, A. J. R. High-sensitivity orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods 9, 1084 (2012).
Kafader, J. O. et al. Measurement of individual ions sharply increases the resolution of orbitrap mass spectra of proteins. Anal. Chem. 91, 2776–2783 (2019).
Contino, N. C. & Jarrold, M. F. Charge detection mass spectrometry for single ions with a limit of detection of 30 charges. Int. J. Mass Spectrom. 345-347, 153–159 (2013).
Kafader, J. O. et al. STORI plots enable accurate tracking of individual ion signals. J. Am. Soc. Mass. Spectrom. 30, 2200–2203 (2019).
Tran, J. C. et al. Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature 480, 254 (2011).
Gómez, S. M., Nishio, J. N., Faull, K. F. & Whitelegge, J. P. The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry. Mol. Cell Proteom. 1, 46–59 (2002).
Smith, R. D. et al. An accurate mass tag strategy for quantitative and high-throughput proteome measurements. Proteomics 2, 513–523 (2002).
The Human Protein Atlas https://www.proteinatlas.org (2019).
Lermyte, F., Tsybin, Y. O., O’Connor, P. B. & Loo, J. A. Top or Middle? Up or Down? toward a standard lexicon for protein top-down and allied mass spectrometry approaches. J. Am. Soc. Mass. Spectrom. 30, 1149–1157 (2019).
Schachner, L. F. et al. Standard proteoforms and their complexes for native mass spectrometry. J. Am. Soc. Mass. Spectrom. 30, 1190–1198 (2019).
Sobott, F. & Robinson, C. V. Characterising electrosprayed biomolecules using tandem-MS—the noncovalent GroEL chaperonin assembly. Int. J. Mass Spectrom. 236, 25–32 (2004).
Valegård, K., Liljas, L., Fridborg, K. & Unge, T. The three-dimensional structure of the bacterial virus MS2. Nature 345, 36–41 (1990).
Asensio, M. A. et al. A selection for assembly reveals that a single amino acid mutant of the bacteriophage MS2 coat protein forms a smaller virus-like particle. Nano Lett. 16, 5944–5950 (2016).
Hartman, E. C. et al. Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle. Nat. Commun. 9, 1385–1385 (2018).
Fornelli, L. et al. Advancing top-down analysis of the human proteome using a benchtop quadrupole-orbitrap mass spectrometer. J. Proteome Res. 16, 609–618 (2017).
Anderson, L. C. et al. Identification and characterization of human proteoforms by top-down LC-21 Tesla FT-ICR mass spectrometry. J. Proteome Res. 16, 1087–1096 (2017).
LeDuc, R. D. et al. Accurate estimation of context-dependent false discovery rates in top-down proteomics. Mol. Cell Proteom. 18, 796–805 (2019).
Ntai, I. et al. Applying label-free quantitation to top down proteomics. Anal. Chem. 86, 4961–4968 (2014).
Durbin, K. R., Skinner, O. S., Fellers, R. T. & Kelleher, N. L. Analyzing internal fragmentation of electrosprayed ubiquitin ions during beam-type collisional dissociation. J. Am. Soc. Mass. Spectrom. 26, 782–787 (2015).
Freeke, J., Robinson, C. V. & Ruotolo, B. T. Residual counter ions can stabilise a large protein complex in the gas phase. Int. J. Mass spectrom. 298, 91–98 (2010).
Skinner, O. S. et al. An informatic framework for decoding protein complexes by top-down mass spectrometry. Nat. Methods 13, 237 (2016).
This work was funded by the Intensifying Innovation program from Thermo Fisher Scientific and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health with additional support from the Sherman Fairchild Foundation, and the instrumentation award (S10OD025194) from NIH Office of Director. In addition, we would like to thank Luca Fornelli and Timothy K. Toby for collecting and analyzing the HEK-293 LC–MS runs utilized for our intact-mass-tag I2MS analysis.
V.Z., A.A.M., J.T.M., D.L.S., P.F.Y. and M.W.S. are employees of Thermo Fisher Scientific.
Peer review information Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Histogram of individual myoglobin +20 ions collected that demonstrate the increased ion survival with decreased Orbitrap central electrode (CE) voltages. The solid blue lines indicate separation of low-intensity noise peaks, mid-level intensity real ions that have decayed during the acquisition event, and high-intensity real ions that did not decay during the detection event. The high intensity ions are used to create an I2MS spectrum. As the CE voltage was reduced from -5 kV to -1 kV, ion velocity decreased and this increased ion survival over a 4 second acquisition from just 3% (CE = -5 kV) to 71% (CE = -1 kV). This finding drastically increased the efficiency of I2MS during its development.
Spectra of a synthetic mixture of intact proteins created by tradition ensemble ion collection plotted in m/z space (a), or via the I2MS process (b). The table in panel (c) lists the known proteins in the mixture along with their masses and corresponding figures of merit determined from the I2MS spectrum in panel (b). The number assigned to each protein in the table correspond to the labeled features in the m/z spectrum and corresponding I2MS spectrum in panels (a) and (b), respectively. Source data
Heavy and Light chain of a monoclonal IgG measured by conventional ensemble FT-MS analysis (a) compared to I2MS (b). Various forms of the Heavy and Light chain species including guanidine adducts and glycosylations caused a rise in baseline in the ensemble spectra were easily distinguished by I2MS and caused no such rise in spectral baseline. Blue arrows are indicative of approximately a 60 Da guanidine adduct and red arrows indicate the addition of a 162 Da hexose-type glycosylation. Peaks arising from assignment of +1 or -1 charge state to individual ions are labeled in the I2MS spectrum with an asterisk*. Source data
Ensemble ion spectra plotted in m/z space (a,d) and their corresponding I2MS mass spectra (b,e) for the ~232 kDa pyruvate kinase (left) and 801 kDa groEL (right) complexes with labeled charge states in panels (a) and (d). The implementation of I2MS yields accurate spectra in the mass domain with resolution (Res) comparable to the various charge states collected in m/z space. Charge state values and relative charge state intensity assignments (c,f) agreed on average within <5% with their corresponding m/z-domain spectra (a,d). Source data
Characterization of the capsid coat proteins (CPs) used to assemble MS2 virus-like particles analyzed in Fig. 3. The WT (top) and MINI (bottom) CP masses are within 4 part-per-million of their theoretical values. Mass measurements from denaturing the VLP capsids produced intact mass values that match and confirm the sequence differences of each species. The one differing amino acid (proline vs. serine at position 37) between the two proteoforms results in the structural differences between the WT and MINI VLPs.
Individual Ion STORI plots and their slope values for +13 (black), +25 (red), and +39 (blue) charge states of ubiquitin, myoglobin, and carbonic anhydrase, respectively. The slope of the Selective Temporal Overview of Resonant Ion (STORI) plot of an individual ion on the outer electrode of the Orbitrap is proportional to the charge of the ion signal. The STORI slope is calibrated externally to provide a single, integer result. See reference10 for a complete description of the STORI process and Fig. 1 for an overview of the entire I2MS process.
Linear calibration of STORI slope values as a function of charge state. Calibration is utilized to determine the charge of an unknown individual ion signal with a calculated STORI slope. The rigorously linear statistical relationship between individual ion STORI slopes and quantized charge states independent of ion m/z is the foundation I2MS is built upon. The proteins utilized to produce the various charge states are labeled by their respective points on the graph. The highlighted red points/trace correspond to the calibration function on a Q-Exactive Plus instrument while the blue points/trace correspond to the calibration function on a Q-Exactive UHMR instrument.
Correct assignment of known +20 myoglobin single ions. As a larger number STORI slope values are averaged prior to the assignment of an ion’s charge (top to bottom) the percentage of ions assigned to the correct (+20) charge state increases markedly. Other examples across the range of charge states used in this study give results from 94.0% to 98.5% in the rate of correct charge states for knowns analyzed by I2MS in either denatured or native modes.
The ensemble spectrum in the m/z domain (a), I2MS mass spectrum (b), and corresponding individual ion charge assignment histogram (c) for the myoglobin charge state envelope. The insets in the m/z spectrum and I2MS mass spectrum demonstrate that although the charges have been condensed in the I2MS spectrum, the features within each charge state remain unchanged. Source data
Supplementary Figures 1–9, Supplementary Protocol and Supplementary Table 1
Software and demo examples
HEK-293 I2 MS Proteoform Assignments; GELFrEE HEK-293 I2MS mass assignments (Fig. 2) with identified proteoform mass, TDReport ID no., PFR no., accession no., modifications and PPM error
Source data for Fig. 2
Source data for Fig. 3
Source data for Supplementary Fig. 2
Source data for Supplementary Fig.3
Source data for Supplementary Fig.4
Source data for Supplementary Fig.9
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Kafader, J.O., Melani, R.D., Durbin, K.R. et al. Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes. Nat Methods 17, 391–394 (2020). https://doi.org/10.1038/s41592-020-0764-5
Trends in Biotechnology (2020)