Online nanoflow strong cation exchange chromatography coupled to native mass spectrometry (nanoSCX-nMS) was introduced in 2024 as a sensitive method for separating intact proteoforms from crude cell lysates under non-denaturing conditions, but the authors noted three limitations in passing: biased loss of acidic (low-pI) proteins on the trap column, degradation of chromatographic resolution when the final elution pH exceeds about 9 due to non-linear pH gradients, and the m/zm/z ceiling (~8000) of the tested mass spectrometer that limits access to very large assemblies whose charge-state envelopes extend beyond it; in addition, the detected species were reported only as deconvoluted intact masses, without sequence-level identification. The low-pI bias strand has since been substantially resolved by the same group's parallel strong-anion/strong-cation exchange platform (Proteomics, 2026), which explicitly leaves volatile-buffer capacity in the pH 8–9.5 window as a persistent challenge, still operates within the 2000–8000 m/zm/z range, and still reports only deconvoluted masses without MS/MS-based identification. The surviving open core is therefore a coherent three-part objective: extend volatile pH-gradient ion-exchange-native MS to very high-pI proteoforms, access very large assemblies whose charge-state envelopes extend beyond the current m/zm/z ceiling, and couple nanoflow intact-mass profiling to confident sequence-level proteoform identification in complex biological samples, building on the unbiased acidic-protein recovery already demonstrated by the dual-exchange platform within its pH 2.6–8.5 gradient windows.

Background

A proteoform is a specific molecular form of a protein arising from its coding sequence combined with post-translational modifications, sequence variants, alternative splicing, or assembly into complexes; different proteoforms of the same gene can have distinct functions. Bottom-up proteomics, the standard workflow in which proteins are digested into peptides before mass spectrometry, cannot directly enumerate proteoforms because it infers proteins from peptide fragments. Top-down proteomics instead measures intact proteins by mass spectrometry, and native mass spectrometry (nMS) does so from non-denaturing solutions (typically volatile ammonium acetate buffers), so that proteins and non-covalent complexes arrive at the detector with folded structures, few charges, and narrow charge-state envelopes — the set of ion peaks corresponding to different charge states of the same species. Because native ions carry fewer charges, their mass-to-charge ratio (m/zm/z) values are high, and instrument m/zm/z ceilings become a hard constraint: a mass spectrometer limited to m/z≈8000m/z\approx 8000 cannot record the charge-state envelope of a very large assembly whose envelope extends beyond that ceiling. Since a species appears at several charge states, its neutral mass is recovered mathematically by deconvolution of the envelope rather than read off a single peak.

Liquid chromatography (LC) separation before nMS reduces the crowding of overlapping envelopes in complex samples. Ion-exchange chromatography (IEC) separates proteins by surface charge: strong cation exchange (SCX) retains positively charged (basic) proteins and releases them as the mobile-phase pH approaches their isoelectric point (pI, the pH at which the protein's net charge is zero), while strong anion exchange (SAX) retains negatively charged (acidic) proteins. In a salt-mediated pH gradient, the ammonium acetate (AmAc) concentration rises while pH changes; because the buffer must be volatile to be MS-compatible, only a narrow set of additives is available, and their buffering capacity is uneven across the pH scale — in particular, weak control above pH ~8 causes abrupt, non-linear pH changes that compress the separation window for very high-pI proteins. Nanoflow LC (hundreds of nL/min) improves electrospray ionization efficiency and sensitivity, and a trap-and-elute setup (loading the sample onto a short pre-column) permits large injection volumes — but an SCX trap can discard acidic proteins that are not retained under loading conditions.

In this setting, Zhai, Mavridou, Damian, Mutti, Schoenmakers, and Gargano (Analytical Chemistry 96(22):8880–8885, 2024) introduced the first online nanoflow SCX–nMS (nanoSCX-nMS) method: a pH 5.0→8.5 salt-mediated gradient on capillary SCX columns with a trap, achieving a detection limit of 0.22 pmol for monoclonal antibodies (mAbs), a peak capacity (number of theoretically resolvable peaks in the gradient window) of 82 in 30 min, resolution of ~141 kDa proteoforms differing by only 54 Da, and roughly 140 deconvoluted intact masses (10–140 kDa) from an E. coli crude lysate, with native states largely preserved. The paper notes, in passing, three limitations relevant to comprehensive characterization of complex proteoform mixtures: (i) acidic proteins may be lost when loading the sample on the SCX trap, biasing the method against low-pI species; (ii) a final elution pH above ~9 reduces chromatographic resolution, presumably because the non-linear pH change above pH 7 abruptly alters the proteins' in-solution charge states, so very high-pI analytes are not reliably covered; and (iii) the charge-state envelopes of the lysate proteins approach the tested instrument's maximum m/zm/z of 8000, so larger assemblies, whose charge-state envelopes extend past that maximum, may be undetectable or undeconvolvable. In addition, the ~140 observed species were characterized only as deconvoluted masses — no MS/MS or orthogonal workflow assigned sequence-level identities — leaving the coupling of intact-mass profiling to confident proteoform identification unaddressed.

Problem Statement

Advance online nanoflow ion-exchange chromatography–native mass spectrometry from intact-mass profiling to comprehensive characterization of proteoform mixtures in crude biological samples (e.g., cell lysates). Unbiased recovery of acidic (low-pI) proteins during sample loading — the trap-bias limitation of the 2024 method — has been addressed by the 2026 parallel strong-anion/strong-cation exchange platform over its pH 2.6–5.0 (SAX) and 5.0–8.5 (SCX) gradient windows and provides the baseline on which further development can build; because that platform's gradients stop at pH 8.5 and that platform names the lack of buffer capacity at pH 8–9.5 as a persistent challenge, unbiased coverage across the proteome's full isoelectric-point range is not yet complete. The open strands to be achieved on an MS-coupled nanoflow ion-exchange-native MS platform applied to complex biological samples are therefore: (1) a volatile, MS-compatible pH-gradient elution that remains linear and controllable up to elution pH above ~9, so that chromatographic resolution is maintained, very high-pI proteoforms are reliably separated, and unbiased coverage across the proteome's full pI range is completed on top of the demonstrated acidic-protein recovery; (2) access, within the nanoflow IEC-nMS workflow, to very large protein assemblies whose charge-state envelopes extend beyond the m/z≈8000m/z\approx 8000 ceiling of the mass spectrometers used so far; and (3) confident sequence-level identification of the detected proteoforms in the complex sample itself, so that the deconvoluted intact masses are assigned to specific proteoform identities rather than remaining an unannotated mass list. Each of the three strands stands independently as a sub-goal whose acceptance standard is the completion criterion stated within that strand; resolving any single strand is a partial resolution of this problem and leaves the other two open, while the full objective — comprehensive characterization of proteoform mixtures — is reached only when all three are addressed.

References

  1. Ziran Zhai, Despoina Mavridou, Matteo Damian, Francesco G. Mutti, Peter J. Schoenmakers, Andrea F. G. Gargano (2024). Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry. Analytical Chemistry 96(22):8880-8885. DOI: 10.1021/acs.analchem.4c01760. https://doi.org/10.1021/acs.analchem.4c01760
  2. Ziran Zhai, Hafsa Zakri, Matteo Damian, Francesco G. Mutti, Garry Corthals, Andrea F. G. Gargano (2026). Parallel Analysis of Acidic and Basic Proteoforms in Cell Lysates via Native Cation and Anion Exchange Chromatography—Native Mass Spectrometry. PROTEOMICS 26(7):158-168. DOI: 10.1002/pmic.70134. https://doi.org/10.1002/pmic.70134
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