Proteins
adopt different higher-order structures (HOS) to enable
their unique biological functions. Understanding the complexities
of protein higher-order structures and dynamics requires integrated
approaches, where mass spectrometry (MS) is now positioned to play
a key role. One of those approaches is protein footprinting. Although
the initial demonstration of footprinting was for the HOS determination
of protein/nucleic acid binding, the concept was later adapted to
MS-based protein HOS analysis, through which different covalent labeling
approaches “mark” the solvent accessible surface area
(SASA) of proteins to reflect protein HOS. Hydrogen–deuterium
exchange (HDX), where deuterium in D2O replaces hydrogen
of the backbone amides, is the most common example of footprinting.
Its advantage is that the footprint reflects SASA and hydrogen bonding,
whereas one drawback is the labeling is reversible. Another example
of footprinting is slow irreversible labeling of functional groups
on amino acid side chains by targeted reagents with high specificity,
probing structural changes at selected sites. A third footprinting
approach is by reactions with fast, irreversible labeling species
that are highly reactive and footprint broadly several amino acid
residue side chains on the time scale of submilliseconds. All of these
covalent labeling approaches combine to constitute a problem-solving
toolbox that enables mass spectrometry as a valuable tool for HOS
elucidation. As there has been a growing need for MS-based protein
footprinting in both academia and industry owing to its high throughput
capability, prompt availability, and high spatial resolution, we present
a summary of the history, descriptions, principles, mechanisms, and
applications of these covalent labeling approaches. Moreover, their
applications are highlighted according to the biological questions
they can answer. This review is intended as a tutorial for MS-based
protein HOS elucidation and as a reference for investigators seeking
a MS-based tool to address structural questions in protein science.