Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes


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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Fig. 1: The five-step workflow for the I2MS process.
Fig. 2: Deconvolution of complex proteoform mixtures.
Fig. 3: Mass determination of large native complexes.

Data availability

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. 24 and 9. Additional desired data that support the charge-determination findings of this study are available from the corresponding authors upon request.

Code availability

Custom compiled code associated with the I2MS creation process is available via Supplementary Software or from the corresponding authors upon reasonable request.


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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.

Author information




Data acquisition and analysis was performed by J.O.K. with assistance from R.D.M. and K.R.D. and with additional input from all authors. R.D.M. prepared samples with help from J.O.K., and B.I. and D.T.-E. assembled and purified the MS2 virus-like particles. J.O.K., B.P.E., K.R.D., R.T.F., S.C.B., P.F.Y., M.W.S. and P.D.C. developed models and created programs to analyze the data. K.R.D., V.Z., A.A.M., J.T.M., D.L.S., M.W.S., P.D.C. and N.L.K. provided additional technical support and guidance. N.L.K., P.D.C. and M.W.S. collected funding support and oversaw the project. J.O.K. and N.L.K. wrote the manuscript. All authors critically reviewed the manuscript.

Corresponding authors

Correspondence to Philip D. Compton or Neil L. Kelleher.

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Competing interests

V.Z., A.A.M., J.T.M., D.L.S., P.F.Y. and M.W.S. are employees of Thermo Fisher Scientific.

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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

Supplementary Figure 1 Ion Survival Histograms.

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.

Supplementary Figure 2 4 Protein Mix.

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

Supplementary Figure 3 Reduced Antibody Standard.

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

Supplementary Figure 4 Native Protein Complexes.

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

Supplementary Figure 5 VLP Subunit Analysis.

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.

Supplementary Figure 6 Individual Ion STORI Plots.

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.

Supplementary Figure 7 Charge Calibration Function.

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.

Supplementary Figure 8 STORI Slope Averaging/Charge Assignment.

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.

Supplementary Figure 9 Myoglobin Example.

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 information

Supplementary Information

Supplementary Figures 1–9, Supplementary Protocol and Supplementary Table 1

Reporting Summary

Supplementary Software

Software and demo examples

Supplementary Dataset 1

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

Source Data Fig. 2

Source data for Fig. 2

Source Data Fig. 3

Source data for Fig. 3

Source Data Supplementary Fig. 2

Source data for Supplementary Fig. 2

Source Data Supplementary Fig. 3

Source data for Supplementary Fig.3

Source Data Supplementary Fig. 4

Source data for Supplementary Fig.4

Source Data Supplementary Fig. 9

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).

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