13 research outputs found
Hydrogen Exchange Mass Spectrometry of Proteins at Langmuir Monolayers
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
Use of MALDI-MS Combined with Differential Hydrogen–Deuterium Exchange for Semiautomated Protein Global Conformational Screening
Matrix-assisted
laser desorption/ionization (MALDI) coupled with
a time-of-flight (TOF) mass-spectrometry (MS) detector is acknowledged
to be very useful for analysis of biological molecules. At the same
time, hydrogen–deuterium exchange (HDX) is a well-known technique
for studying protein higher-order structure. However, coupling MALDI
with HDX has been challenging because of undesired back-exchange reactions
during analysis. In this report, we survey an approach that utilizes
MALDI coupled with an automated sample preparation to compare global
conformational changes of proteins under different solution conditions
using differential HDX. A nonaqueous matrix was proposed for MALDI
sample preparation to minimize undesirable back-exchange. An automated
experimental setup based on the use of a liquid-handling robot and
automated data acquisition allowed for tracking protein conformational
changes as a difference in the number of protons exchanged to deuterons
at specified solution conditions. Experimental time points to study
the deuteration-labeling kinetics were obtained in a fully automated
manner. The use of a nonaqueous matrix solution allowed experimental
error to be minimized to within 1% RSD. We applied this newly developed
MALDI-HDX workflow to study the effect of several common excipients
on insulin folding stability. The observed results were corroborated
by literature data and were obtained in a high-throughput and automated
manner. The proposed MALDI-HDX approach can also be applied in a high-throughput
manner for batch-to-batch higher-order structure comparison, as well
as for the optimization of protein chemical modification reactions
Membrane-Associated Conformation of HIV‑1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer
In
the companion paper to this work, we described development of
a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement
that integrates Langmuir monolayers. With Langmuir monolayers, the
lipid packing density can be reproducibly controlled and changed as
desired. Analysis of HX in proteins that may undergo conformational
changes as a function of lipid packing (for example, conformational
rearrangements after insertion into a lipid layer) are then possible.
We previously used neutron reflection to characterize just such a
conformational change in the myristoylated HIV-1 Nef protein (myrNef):
at high lipid packing density, myrNef could not insert into the lipids
and maintained a compact conformation adjacent to the monolayer, whereas
at lower lipid packing density, myrNef was able to insert N-terminal
arm residues, causing displacement of the core domain away from the
monolayer. In order to locate where conformation may have been altered
by lipid association, we applied the HX MS Langmuir monolayer method
to myrNef associated with monolayers of packing densities identical
to those used for the prior neutron reflection measurements. The results
show that the N-terminal region and the C-terminal unstructured loop
undergo conformational changes when associated with a low density
lipid monolayer. The results are not consistent with the hypothesis
of myrNef dimerization upon membrane association in the absence of
other myrNef binding partners. The HX MS Langmuir monolayer method
provides new and meaningful information for myrNef that helps explain
necessary conformational changes required for function at the membrane