>> Hello, and welcome to the next in our OSTR series on cutting-edge methodologies. Today,
we're going to talk about structural mass spectrometry approaches, and specifically,
hydrogen-deuterium exchange mass spec. Hydrogen-deuterium exchange is a methodology for looking at changes
in protein flexibility and protein confirmation. In the method, regions with secondary structure,
such as helices or sheets, tends to have low levels of exchange, whereas loops tend to
have a higher level of exchange. And so by incubating proteins with deuterium, there
will be an exchange between the proton and deuterium and we can use mass spec to try
and look at the difference in uptake of deuterium for protons. If you look at a single peptide
like this, there are several hydrogens that could exchange. Within the side chain, there
tends to be very rapid exchange rate that goes so quickly that it's impossible for us
to detect. In contrast, the aliphatic hydrogen on the carbon doesn't exchange at all, so
that one we're also going to lose. Conveniently, the backbone amide group does have an exchange
rate on the order of milliseconds to days, and so it's these exchange reactions that
we're going to be analyzing. Hydrogen-deuterium exchange can be applied to several different
biological questions. For example, one can ask does a protein fold correctly? So the
difference between different states and different folding forms can be identified by looking
at changes in deuterium uptake. One can also look are there conformational changes after
application of a stress, after addition of a ligand, or changes in some form of a composition
of the buffer or physiological condition. One can also ask where does one protein bind
another? Theoretically, in the binding site, there will be changes in deuterium uptake
that can be used to map binding sites. One can ask can I identify sites of protein aggregation
which would also affect deuterium uptake? One can look at how mutations affect protein
structure, as well as where drugs interact with target proteins. Really, HDX is a nice
methodology to look at protein structural analysis that enables the study of higher
order structure, especially conformational changes in protein binding interactions. There
are several factors that actually affect the HDX itself. These include solvent accessibility
and hydrogen bonding, both of which are factors that report on the structure and confirmation
of the protein. Also, pH and temperature will affect the deuterium uptake and these can
be experimentally controlled. A general HDX mass spec workflow would begin with a protein
alone or a protein plus a ligand. This is then incubated in D2O for a labeling period
during which accessible protons will exchange to deuterium. Next is a very critical denaturation
and quenching step at which the protein is kept at zero degrees in an acidic low pH,
around 2.5. These steps are critical to maintain the deuterium that's on the protein and not
allow back exchange to a proton. After this quenching step, proteolysis occurs using pepsin.
Pepsin and other acidic proteases are the enzymes of choice for this type of analysis
because they are functional at low temperature and low pH. After this proteolysis step, peptides
are separated and then analyzed by mass spectrometry. These analyses can then occur over multiple
different timeframes such that the deuterium uptake over time can be analyzed and one can
look at changes in deuterium chain level in the protein over time.
^M00:03:48 In addition, liquid handling steps are often
used in an automated way to improve reproducibility and maximize instrument throughput. So that
more samples can be analyzed and there's less instrument downtime. There are several programs
that are used for HDX analysis and these are all shown on the screen. Some of these are
publicly available and some of them are fee-based products, and they all have different benefits
and disadvantages, so these are here for you as a resource. All of these methods, though,
will go through the same type of step, which is acquiring data, defining what the protein
is, looking at the mass spec data. Detecting unlabeled peptides, and then using those to
generate an HDX peptide list from the full experiment, detecting those modified peptides.
And then interpreting the data and forming comparisons such that one can start to look
at the effects of whatever treatment is being used. This is an example of the deuterium
uptake map for a specific protein. You can see the sequence as well as different peptides
that have been identified. And those peptides are color-coded by how much deuterium has
been observed to be taken up across different times. A deuterium uptake is based upon mass
shifts due to the change in mass from deuterium to proton. As you can see throughout the protein,
there are different regions with either lower or higher relative deuterium uptake. Those
different peptides can then be analyzed across different times and one can see changes in
the deuterium uptake at different times. In a standard experiment, there is some scrambling
of the deuterium across the peptide such that it's hard to have single amino acid resolution.
Methods using ETD for fragmentation do tend to maintain the localization of the deuterium
on the residue. In addition, one can look at peptides with overlapping sequences to
try and get more information about where the deuterium change is. So by looking at those
overlapping peptides, for example here, more information about the exchange can be obtained.
So you can see that in the shorter peptide, they're slightly higher uptake than in the
longer peptide, suggesting that there is a difference overall from the N terminal to
the C terminal part of the peptide. In this example, the authors used deuterium exchange
to identify the site of antigen binding on an antibody. So here, the antigen was bound
by the monoclonal antibody and you can see protected regions when the antigen bound are
highlighted in red. And these were identified based on changes in the deuterium uptake.
So this is just an example of how one can use the process to map changes in protein
confirmation and try and identify where ligands are binding, where effects are taking place.
These are very interesting studies that could be well applicable to different confirmational
methods. So if you are interested I suggest you talk to your local mass spectrometrist.
Within the NCI, you can talk to me, Lisa Jenkins, or my colleague, Thorkell Andresson, in Frederick.
Within the wider NIH community, I would direct you to visit the CREx for more information
about available resources. Thank you very much for your attention.
>> U.S. Department of Health and Human Services, National Institutes of Health, National Cancer
Institute, Cancer.gov, 1-800-4-Cancer. Produced August 2019.