Hydrogen Exchange Mass Spectrometry of Proteins at
Langmuir Monolayers
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Abstract
Hydrogen
exchange (HX) mass spectrometry (MS) is valuable for providing
conformational information for proteins/peptides that are very difficult
to analyze with other methods such as peripheral membrane proteins
and peptides that interact with membranes. We developed a new type
of HX MS measurement that integrates Langmuir monolayers. A lipid
monolayer was generated, a peptide or protein associated with it,
and then the monolayer-associated peptide or protein was exposed to
deuterium. The deuterated species was recovered from the monolayer,
digested, and deuterium incorporation monitored by MS. Test peptides
showed that deuterium recovery in an optimized protocol was equivalent
to deuterium recovery in conventional solution HX MS. The reproducibility
of the measurements was high, despite the requirement of generating
a new monolayer for each deuterium labeling time. We validated that
known conformational changes in the presence of a monolayer/membrane
could be observed with the peptide melittin and the myristoylated
protein Arf-1. Results in an accompanying paper show that the method
can reveal details of conformational changes in a protein (HIV-1 Nef),
which adopts a different conformation, depending on whether or not
it is able to insert into the lipid layer. Overall, the HX MS Langmuir
monolayer method provided new and meaningful conformational information
for proteins that associate with lipid layers. The combination of
HX MS results with neutron or X-ray reflection of the same proteins
in Langmuir monolayers can be more informative than the isolated use
of either method