>> Hi, welcome to the next in our OSTRD Series on cutting edge methodologies. Today, we're
going to talk about proximity dependent biotinylation for the characterization of protein complexes
and the mapping of organelles. A standard experiment for characterization of protein
protein complexes involves pull down of a base coupled with mass spectrometry to identify
the prey that copurifies with it. This is a mass spec variant of a standard IP or pull
down experiment. These are nice experiments, they're relatively straightforward, routinely
used, if one can do an IP Western adding it into a mass spec detection isn't very difficult.
And they're also readily coupled to quantitative methods to get relative information on changes
in enrichment. However, there are some disadvantages. For example, you can lose interactions where
an antibody binds if you're doing amino precipitation. The antibody and protein interactors are going
to compete for binding to the same sites and the antibody is generally going to win. These
experiments don't work well for hard to purify interactors, those that are transient or form
unstable complexes. Also, for enzymes, because the substrates of these enzymes are often
low affinity interactions, they're meant to be released and not stable. And also, for
baits that have poor solubility such as proteins from membranes. Standard IP mass spec experiments
are also subject to post-lysis artifacts. These are interactions that form only in the
life state; they don't normally form under physiological conditions between 2 proteins
that aren't normally close together in a cell. So, there are new ways that have been developed
to try and overcome some of these disadvantage is and help to find better protein complex
information. The class of experiment that we're going to talk about today is proximity-dependent
biotinylation or PDB experiments. In these experiments, there's expression of a fusion
protein of the bait with an enzyme that catalyzes activation of biotin or phenolic biotin derivative.
These are either going to be biotin ligases or peroxidases. Expression of the bait fusion
with the relevant context and using a biotin substrate leads to biotinylation of proteins
that are close by in the cell. Lysis of the cell can then happen under potentially harsh
conditions, but you can get full solubilization of membrane proteins and capture of biotinylated
proteins by streptavidin pull down. This means that unstable or transient complexes could
still be tagged and are still able to be pulled down and so they can find interactors regardless
of their affinity or their presence in the membrane. So, let's look at some of these
examples. Biotin ligases are commonly used such as the BirA or BioID tags. In these methods,
they use activation of biotin to biotin oil AMP. These enzymes have been modified so they're
unable to catalyze direct transfer to a specific residue but instead they release a cloud of
activated biotin which reacts with epsilon amines on nearby proteins within approximately
10 nanometers of labeling radius. And the other side is peroxidases such as the Apex
and Apex 2 enzymes which catalyzed tyrosine attack by phenolic compounds which are coupled
to biotin which are activated by peroxide. These have a slightly larger labeling radius
of about 20 nanometers. These are similar though so the major difference in the 2 methods
is the time required for sufficient signal for mass spec analysis. BioID experiments
are generally performed with labeling of 12 to 24 hours, although the new turbo ID and
many turbo tags can generate strong signals reportedly in minutes. In contrast, the Apex
2 methods which use peroxide are definitely performed on the time scale of minutes, so
really the approach to go with depends on the type of experiment and what type of labeling
speed you need. Just to go again into an example as you can see on this slide, what you have
is either BioID or Apex where there are proteins in complex. When they're activated, those
proximal proteins get biotinylated and that can then be pulled down on streptavidin sepharose
for mass spectrometry analysis. Since these streptavidin biotin interactions is so tight,
these experiments can be performed under harsh to denaturing conditions for the cell lysis
and also understand stringent washing conditions to help bring down the background. Looking
here at the cell, you can see again this is the scale in which things happen. The nucleolus
is on the scale of 2 to 3 microns so that when we're getting labeling radii of 10 to
20 nanometers this really is a small labeling radius and so we can get good modification
within protein complexes or within organelles. There are some, of course, important controls
and things to consider with these types of experiments just as with any experiment. First
is the background. There are, of course, natively biotinylated proteins in the cell so one has
to consider the ability to identify those as well. They're also going to be proteins
that are promiscuously biotinylated due to some mislocalization of the fusion protein
as well as proteins that will nonspecifically interact with the resin. So, because of these
proteins that contribute to the background, controls are very important. Controls to determine
endogenous biotinylations such as the bait protein without addition of the enzyme or
untransfected cells are often useful. Controls of the conditions that look at promiscuous
biotinylation are also helpful. In this case, that would be just the enzyme alone or effused
to an irrelevant protein that's unrelated to what you're studying. And furthermore,
replicate analyses really help. These background proteins are going to tend to be low or random
presence so that when doing replicate experiments true interactors should come out time and
time again. There are also some caveats to these experiments that are important to keep
in mind. The residence time of proteins in the complex and the number of solvent-exposed
reactive residues will somewhat determine how detectable proteins can be. Labeling also
depends on the protein and complex side. So, for example, if you have a very large complex,
not all members will be labeled by a single bait and so multiple different baits may be
required. Further, for bait proteins that are embedded in membranes or localized to
organelles, abundant proteins that are present in those locations but are not necessarily
direct interactors will likely still be present and may dominate that identification list.
Just because there are a lot of them, they're going to tend to be able to be labeled. So,
in that case, an extra experimental control in which proteins with the same localization
are used but different interactors or even a mutant form of the bait protein may help
to distinguish those possibilities. So, this is an example using BioID where the authors
were using this method to look at proteins associating with mRNA biology looking at specifically
proteins in RNA granules either stress granules or processing bodies. Using the methodology,
they were able to find over 7,000 unique interactions with nearly 1800 proteins. Analysis of the
correlated patterns between the preys uncovered spatial organization of RNA regulatory structures
and enabled definition of core components of both the stress granules and the processing
bodies. So, the method here was really applied nicely to these different subcellular localization
groups and able to identify what actually constitutes the different bodies. In contrast,
this is an example using Apex to study g-protein coupled receptor signaling. First, the authors
established that the Apex method could be used to characterize complexes of their GPCR
after treatment with the agonist and with the antagonist and identified approximately
1200 protein interactors in the agonists-treated cells including small g-proteins. None of
those interactors were enriched in the antagonist-treated cells. So, knowing that the method worked,
they could then use it to do a time-resolved experiment where they were able to track the
interactions at 10 different time points using 10-second intervals for the first 50 seconds
and then longer intervals up to 30 minutes. In addition, they had one sample with no ligand
treatment at all as a control. And so, using the Apex method and the fact that it works
over very short time scales, they were able to track proximity kinetics for over 1,000
proteins. They were able to look at enzycytosis and subcellular localization over longer time
scales looking at the changes in the interactors known to be involved in the process or in
the localization. And so, this is a very unique experiment to be able to look at the effects
of protein signaling complexes over time, look at how the complexes form and fall apart
and it's really a method that can only be done with this type of approach and experimental
design. So it's just something to think about when planning your experiment to talk to someone
to ask what their preferences are and what methods could be best applied to your own
experiments. And for that I would suggest in the NCI intramural program you can always
talk to me, Lisa Jenkins or my colleague Thorkell Andresson up in Frederick and we would be
happy to discuss with you about your phosophopeptide needs and your analysis desires. Within the
NIH community you can also visit the CREx for more information about available resources.
Thank you very much for your time attention.
So, for more information about these types of methods and to think about how you could
use them in your own research, please feel free to contact your local mass spectrometerist.
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 [inaudible] 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 1-800-4CANCER. Produced August 2019.