Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes

With an overarching goal of characterizing the structure of every
protein within a cell, identifying its interacting partners, and quantifying
the dynamics of the states in which it exists, key developments are
still necessary to achieve comprehensive native proteomics by mass
spectrometry (MS). In practice, much work remains to optimize reliable
online separation methods that are compatible with native MS and improve
tandem MS (MS/MS) approaches with respect to when and how energy is
deposited into proteins of interest. Herein, we utilize native capillary
zone electrophoresis coupled with MS to characterize the proteoforms
in the Escherichia coli 70S ribosome.
The capabilities of 193 nm ultraviolet photodissociation (UVPD) to
yield informative backbone sequence ions are compared to those of
higher-energy collisional dissociation (HCD). To further improve sequence
coverage values, a multistage MS/MS approach is implemented involving
front-end collisional activation to disassemble protein complexes
into constituent subunits that are subsequently individually isolated
and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique
proteoforms, including 22 protein–metal complexes and 10 protein–protein
complexes. Additionally, mapping metal-bound holo fragment ions resulting
from UVPD of protein–metal complexes offers insight into the
metal-binding sites.