Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes - PubMed (original) (raw)

. 2020 Apr;17(4):391-394.

doi: 10.1038/s41592-020-0764-5. Epub 2020 Mar 2.

Rafael D Melani 1, Kenneth R Durbin 1, Bon Ikwuagwu 2, Bryan P Early 1, Ryan T Fellers 1, Steven C Beu 3, Vlad Zabrouskov 4, Alexander A Makarov 5, Joshua T Maze 6, Deven L Shinholt 6, Ping F Yip 4, Danielle Tullman-Ercek 2, Michael W Senko 4, Philip D Compton 7, Neil L Kelleher 8

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

Jared O Kafader et al. Nat Methods. 2020 Apr.

Abstract

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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Conflict of interest statement

COI Statement

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

Figures

Figure 1.

Figure 1.. The five step workflow for the I2MS process.

Includes data acquisition and post-acquisition processing steps that culminate in the plotting of an I2MS spectrum (Step 5). A detailed explanation of each of these five steps is found in the accompanying Online Methods section.

Figure 2.

Figure 2.. Deconvolution of Complex Proteoform Mixtures.

Comparison of the number of proteoform assignments possible using either conventional spectral acquisition with an ensemble of ions in a population (a) or the I2MS process (b). This comparative analysis involved directly infusing a mixture of 0 – 30 kDa proteins created by fractionation of whole extracts from HEK-293 human cells. The inset in panel (a) shows an analytical, silver-stained gel, with the fraction analyzed highlighted with a red rectangle. The 3 insets in panel (b) highlight 17 of the 550 total proteoforms assigned, which are listed in Supplemental Table 1 and include their proteoform record numbers (PFRs). The 20–25 kDa region highlighted with a red rectangle in panel (b) corresponds to proteoforms of higher mass proteins in the complex mixture previously unidentified using LC-MS/MS for top-down proteomics.

Figure 3.

Figure 3.. Mass Determination of Large Native Complexes.

Crystal structure renderings (a,b) and mass spectra of assembled WT (c,e,g) vs. MINI (d,f,h) MS2 virus-like particles, with approximate masses and capsid diameters of 3.2 vs. 1 MDa and 27 vs.17 nm, respectively. In panels (c) and (d), the standard MS spectra plotted in m/z space results in a loss of charge state resolution due to heterogeneity of cargo inside the capsid of the virus-like particles. However, the corresponding I2MS spectra in panels (e) and (f) allow mass assignment to the distribution of particles even without resolution of the individual charge states (a normal requirement of conventional data produced by electrospray MS). The individual ions assigned to their underlying charge distributions are shown in panels (g) and (h). Mass and resolution (Res.) at full-width of the half-maximum (FWHM) of the peak heights are labeled beside each peak.

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