Figures
Figure 1 : Characterization of proteins using denaturing CZE conditions and denaturing ESI-MS as well as native CZE, native CIEF and ACE coupled to native MS, respectively.
Figure 2 : Representation of the migration of cations in a CE-MS setup depending on capillary coating, the resulting EOF and for the required polarity. (A) shows the migration in an uncoated, (B) in a neutrally coated capillary and (C) using a cationic (SMIL) capillary coating. The length of the arrows represents the strength of the EOF (green), the magnitude of analyte mobility (orange) and the magnitude of the resulting apparent mobility (blue).
Figure 3. Evaluation of BGE composition on separation performance of native CZE-MS. (A) Base peak electropherograms for SigmaMab with 10 mM AmAc (AA), 10 mM AmF (AF), and 50 mM AmAc as the BGE. The peaks labeled with black boxes in the electropherograms represent the same mAb species. (B) Extracted ion electropherograms (EIEs) of four different model proteins separated using 20, 40, and 60 mM AmAc as BGE, respectively. Separation improved with increasing AmAc concentration. EIEs for the proteins were created using the three highest charge states, respectively. Adapted and reprinted with permission from ref (A) and (B). Copyright © 2021, Elsevier.
Figure 4. (A) Base peak electropherogram of E. coliribosomal proteins with 500 μM magnesium acetate in the BGE. (B) Corresponding mass spectra of the dominant species in (A) collected at (1) 53.72 and (2) 65.41 min allowed these species to be identified as the 30S and 50S subunits. Base peak electropherograms of subcomplexes and single proteins after rRNA removal with (C) no Mg2+ and (D) 100 μM Mg2+ in the BGE. Reprinted with permission from ref . Copyright © 2020 ACS, American Chemical Society.
Figure 5. EIEs of a sample containing H2BK120ub and H3K4,9,14,18ac analyzed with (a) standard (5 psi supplemental pressure) and (b) high-resolution (2.2 psi supplemental pressure) CZE methods. c−f: corresponding mass spectra generated by averaging the EIE peaks. Reprinted with permission from ref . Copyright © 2021, American Chemical Society.
Figure 6. Example for an ACE-nMS application showing a sheathless CE−MS separation obtained for a H2O2-stressed (A) NGmAb or (B) AAA-mAb sample using a BGE without FcRn (upper panel) and containing FcRn (lower panel). The blue trace corresponds to the extracted ion electropherograms (EIEs) of the double-oxidized /two red stars), the green trace corresponds to the EIEs of mono-oxidized (one red star), and the brown trace shows the EIEs of the non-oxidized mAbs present in the mixture. Signal intensities are normalized. Reprinted with permission from . Copyright © 2021 The Authors. Published by American Chemical Society. Licenced under CC BY-NC-ND 4.0.
URL: https://pubs.acs.org/doi/10.1021/acs.analchem.1c03560.
Figure 7. Example for a nCIEF-nMS application. (A) Base peak electropherogram of streptavidin (SA) analyzed by nCIEF-MS; (B) deconvoluted mass spectra of seven SA charge variants separated from nCIEF-MS. The inserted figure shows the zoomed-in EIEs for the overlapping variants. Adapted and reprinted with permission from . Copyright © 2022, American Chemical Society.
Table 1: Analysis of proteins using native CZE-MS, including MCE-MS and ACE-MS.