Development of a Nitric Oxide-Responsive Labeling Reagent for Proteome Analysis of Live Cells

Nitric
oxide (NO) is a pleiotropic signaling molecule involved
in the regulation of diverse physiological and pathophysiological
mechanisms in cardiovascular, nervous, and immunological systems.
To understand the biological functions of NO in detail, comprehensive
characterization of proteins found in high-NO concentration environments
is crucial. Herein, we describe the design of NO-responsive protein
labeling reagents based on N-alkoxyacyl-o-phenylenediamine as an optimal reactive scaffold. The designed molecules
can label proteins in murine macrophage cells in response to endogenously
produced NO. The combination of NO-responsive protein labeling and
liquid chromatography–tandem mass spectrometry technology allowed
the characterization of the proteome under NO-generated conditions.
Moreover, we demonstrated that our reagent was able to selectively
mark and be used to fluorescently visualize NO-producing cells in
a mixed cell culture system.