Speaker
Description
Top-down proteomics is a powerful tool for investigating complex biological processes in living organisms, both in health and disease. Unlike the more common bottom-up approach, top-down proteomics does not involve digesting proteins into peptides, providing a more comprehensive view of the proteome landscape. However, using intact proteins instead of peptides presents experimental challenges such as reduced sensitivity, low fragmentation efficiency, and difficulties in generating isotopically resolved precursor and product ion spectra.
Charge Detection Mass Spectrometry (CD-MS) is a single-ion technique where the ion's charge is directly inferred from its image current for each individual ion, eliminating the need for resolvable m/z features in ensemble mass spectra. While CD-MS has primarily been used for high-mass native applications, where individual charge states are not resolvable due to the heterogeneous nature of large protein complexes, its benefits can also be applied to (native) top-down proteomics.
CD-MS offers increased sensitivity and the ability to deconvolve congested spectra, ensuring that each peak corresponds to a single protein or peptide ion. This is particularly advantageous for top-down applications, where spectra are typically complex and congested. Thermo Scientific™ Direct Mass Technology™ mode, one of the few commercially available CD-MS solutions, has demonstrated its capability in improving the detection of low-abundant proteoforms (DOI:0.1021/acs.jproteome.9b00797) and achieving isotopic resolution for high-m/z native complexes (DOI: 10.1021/acs.analchem.0c03282).
In this study, we compare the performance of the standard and high-field Orbitrap™ mass analyzers using the Direct Mass Technology mode on native protein complexes. We explore their differences at longer transient times (4 seconds) and demonstrate the increased mass and charge accuracy of the more compact high-field Orbitrap mass analyzers.
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