Multinotch Isolation Enables Efficient Native Top-down Mass Spectrometry of Proteins in Complex Mixtures.
- 1School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom, B15 2TT
Abstract
Accurate identification of intact proteoforms and their complexes directly from heterogenous biological samples is essential to understand their role in health and disease. While native top-down mass spectrometry (nTDMS) workflows enable this, the extreme molecular complexity of biological samples inadvertently results in overlapping signals which confounds protein identification. One approach to resolving this complexity is gas-phase ion-ion reactions which separate analytical targets according to their original charge state. Charge reduction techniques coupled to mass spectrometry resolve signal overlap in m/z space, when ions differ in charge, and so they are especially useful in the analysis of large biomolecules like proteins that form multiple charge states during electrospray ionisation. Previously, we investigated multistage nTDMS of intact proteins sampled directly from tissue sections where the first experiment stage was separation by charge reduction, followed by collisional activation for sequence ion production. The specificity of product ion spectra was improved but at the expense of signal intensity owing to inefficient use of charge-reduced product ions. Here we establish the use of synchronous precursor selection (SPS) for nTDMS via multinotch isolation in a linear ion trap, enabling simultaneous isolation of multiple targets in m/z space and thereby increasing the number of ions available for fragmentation. We implemented SPS for nTDMS across three charge reduction techniques and collision, electron and photon mediated fragmentation methods on commercial and modified mass spectrometers. Improvements to the signal-to-noise ratio of product ions resulted in greater sequence coverage for example intact proteins and complexes up to ~44 kDa using the SPS workflow. We provide a freely-available SPS target calculator, Novalap, that enables simple SPS method development. SPS nTDMS is a methodological breakthrough for proteoform identification in complex mixtures, where specificity is essential, sequence coverage is desired and ion abundance is at a premium.
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BibTeX
@misc{hale2026multinotch,
title = {{Multinotch Isolation Enables Efficient Native Top-down Mass Spectrometry of Proteins in Complex Mixtures.}},
author = {Oliver J. Hale and Helen J. Cooper},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010147/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010147/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Multinotch Isolation Enables Efficient Native Top-down Mass Spectrometry of Proteins in Complex Mixtures.
AU - Oliver J. Hale
AU - Helen J. Cooper
PY - 2026
DA - 2026/10/09
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010147/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010147/v1
AB - Accurate identification of intact proteoforms and their complexes directly from heterogenous biological samples is essential to understand their role in health and disease. While native top-down mass spectrometry (nTDMS) workflows enable this, the extreme molecular complexity of biological samples inadvertently results in overlapping signals which confounds protein identification. One approach to resolving this complexity is gas-phase ion-ion reactions which separate analytical targets according to their original charge state. Charge reduction techniques coupled to mass spectrometry resolve signal overlap in m/z space, when ions differ in charge, and so they are especially useful in the analysis of large biomolecules like proteins that form multiple charge states during electrospray ionisation. Previously, we investigated multistage nTDMS of intact proteins sampled directly from tissue sections where the first experiment stage was separation by charge reduction, followed by collisional activation for sequence ion production. The specificity of product ion spectra was improved but at the expense of signal intensity owing to inefficient use of charge-reduced product ions. Here we establish the use of synchronous precursor selection (SPS) for nTDMS via multinotch isolation in a linear ion trap, enabling simultaneous isolation of multiple targets in m/z space and thereby increasing the number of ions available for fragmentation. We implemented SPS for nTDMS across three charge reduction techniques and collision, electron and photon mediated fragmentation methods on commercial and modified mass spectrometers. Improvements to the signal-to-noise ratio of product ions resulted in greater sequence coverage for example intact proteins and complexes up to ~44 kDa using the SPS workflow. We provide a freely-available SPS target calculator, Novalap, that enables simple SPS method development. SPS nTDMS is a methodological breakthrough for proteoform identification in complex mixtures, where specificity is essential, sequence coverage is desired and ion abundance is at a premium.
ER -