Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures

Native top-down mass spectrometry
(nTDMS) allows characterization
of protein structure and noncovalent interactions with simultaneous
sequence mapping and proteoform characterization. The majority of
nTDMS studies utilize purified recombinant proteins, with significant
challenges hindering application to endogenous systems. To perform
native top-down proteomics (nTDP), where endogenous proteins from
complex biological systems are analyzed by nTDMS, it is essential
to separate proteins under nondenaturing conditions. However, it remains
difficult to achieve high resolution with MS-compatible online chromatography
while preserving protein tertiary structure and noncovalent interactions.
Herein, we report the use of online mixed-bed ion exchange chromatography
(IEC) to enable separation of endogenous proteins from complex mixtures
under nondenaturing conditions, preserving noncovalent interactions
for nTDP analysis. We have successfully detected large proteins (>146
kDa) and identified endogenous metal-binding and oligomeric protein
complexes in human heart tissue lysate. The use of a mixed-bed stationary
phase allowed retention and elution of proteins over a wide range
of isoelectric points without altering the sample or mobile phase
pH. Overall, our method provides a simple online IEC-MS platform that
can effectively separate proteins from complex mixtures under nondenaturing
conditions and preserve higher-order structure for nTDP applications.