Enhancing Top-Down Characterization of Membrane Proteoforms with C8-Functional Amine-Bridged Hybrid Monoliths

Comprehensive characterization of membrane proteins at
the level
of proteoforms in complex biological samples by top-down mass spectrometry
(MS) is of vital importance in revealing their precise functions.
However, severe peak broadening in the separation of hydrophobic membrane
proteins, caused by resistance to mass transfer and strong adsorption
on separation materials, leads to MS spectra overlap and signal suppression,
which makes against the in-depth research on membrane proteoforms.
Herein, C8-functional amine-bridged hybrid monoliths with an interconnected
macroporous structure were developed by the in situ one-step sol–gel
reaction of triethoxy(octyl)silane and bis[3-(trimethoxysilyl)propyl]amine
in capillaries. Due to the unique macroporous structure and bridged
secondary amino groups in the framework, the monolith possessed reduced
resistance to mass transfer, low nonspecific adsorption, and electrostatic
repulsion to membrane proteins. These features tremendously alleviated
peak broadening in the separation of membrane proteins, thus outperforming
traditional reversed-phase columns in top-down characterization of
membrane proteoforms. With this monolith, a total of 3100 membrane
proteoforms were identified in the mouse hippocampus, representing
the largest membrane proteoform database obtained by top-down analysis
so far. The identified membrane proteoforms revealed abundant information,
including combinatorial post-translational modifications (PTMs), truncation,
and transmembrane domains. Furthermore, the proteoform information
was integrated into the interaction network of membrane protein complexes
involved in oxidative phosphorylation processing, opening up new opportunities
to uncover more detailed molecular basis and interaction in the biological
processes.