>> Hi, welcome to the next in our OSTR Series on applications and methodology. Today we're
going to talk about analysis of post-translational modifications by mass spectrometry and specifically
analysis of phosphorylation. Post-translational modifications are covalent processing events
that change the properties of an amino acid or a protein. Two important aspects in the
study of post-translational modifications are identification of the site of modification
and understanding of how the change in that site affects the functional consequences for
the protein. Those consequences include activation of a protein, changes in its localization
or stability, or changes in protein-protein interactions. There are lots of different
types of post-translational modifications and they include addition of functional groups
such as you see here, alkylation acylation, oxidation and phosphorylation, also additions
of proteins or peptides, these include ubiquitination, sumoylation, and neddylation, changes in the
chemical nature of amino acids such as citrullination and deamidation as well as changes in the
structure of an amino acid or protein such as the disuflide bridges, proteolytic cleavage
and racemization of prolines. Post-translational modification is not necessarily a straightforward
process. The same amino acid can be modified in multiple ways. For example, the same lysine
residue could have an acetylation, a ubiquitous modification or a methylation modification.
These will each compete with one another and each can have different effects for the protein
itself. So, in the area of proteomics, how do we study these modifications? We know that
there are critical changes in the proteome and protein regulation that occur during development
following the stress or during disease states. For example, all stages of the frog development
have the same genome, but the proteome protein regulation and pro-translational modifications
all differ. Generally, when we're looking at modifications, we want to start with one
of two things. We're going to do an enrichment either at the protein level or at the peptide
level. So, we can either do the enrichment first followed by a digestion step and then
detection of peptides or we could do the digestion first, enrich the peptides we're interested
in and then finally detect them again. So, if we focus specifically on phosphorylation,
there are several ways to do this. First would be antibody-based methods for enrichment.
This can be used to apply to both proteins and peptides, but you need a high-quality
antibody with strong antigenicity for the pulldown. There are also charge-based methods
that are used for phosphopeptides as well as protein interactor-based methods which
could be used at both the protein or peptide level and are generally used for phosphotyrosine.
This is an example of the use of antibodies to pull down phosphopeptides. In this case,
the different cells were grown in different media for quantitation and then the peptides
were mixed, digested with trypsin and then the phosphopeptides were immune precipitated
with different antibodies for different specific phosphorylation sites. After that enrichment,
mass spectrometry analysis was performed for both the identification and quantification
of changes in protein and peptide level. Using this method, peptides with 68 different anti-phospho
antibodies were used. The authors were able to identify almost a 1,000 regulated phospho
sites and 700 proteins and they found 55 regulated sites on 31 candidate substrates, so this
is just a methodology showing that different antibodies can be used to pull off different
overlapping sites not just the site for which they're specifically raised. Charge-based
methods also work very well to pull out phosphopeptides and are very commonly used. In this case,
you have some sort of a charged group on the bead which is used to pull out the negatively
charged phosphate group on the peptide. The group on the bead could be an IMAC; this is
an immobilized metal affinity chromatography, in which case it's usually iron or galleon
that's attached to the bead. You can also do titanium or zirconium oxide and there's
also been a product that's developed called a phos tag which is a specific group that
will be attached to an [inaudible] bead. Researchers have also used sequential use of the IMAC
and titanium dioxide for better separation of different phosphorylated sites. So, let's
look at a few examples. So, in this case, the authors are using either titanium, zirconium
or IMAC to pull out different peptides. And what they found was that the titanium had
the highest number of phosphopeptides and the ratio of phospho to non-phosphopeptides,
depending on the source, there was different retentivity and lower selectivity. They found
the IMAC to be less retention but had selectivity similar to that of titanium dioxide. In another
example, they compared phosphoramidite chemistry, IMAC and titanium dioxide. They found similar
characteristics among all of them and they found that no one method was more comprehensive
than any other. As you can see in the Venn diagram, each had overlapping identifications
but there were also some phosphopeptides that were only identified in one of the different
methods. Each method again identified different partially overlapping phosphopeptides, so
a methodology that used complements of all of these might be the best approach. This
is an example for the sequential use of IMAC and titanium dioxide. In this case, they found
that the sequential application was better than titanium dioxide alone and they found
that the sequential use of these methods was better for enriching mono-phosphorylated versus
di- and tri-phosphorylated peptides. So again, depending on application, the methodology
could be differed, and they may be complementary results. One of my favorite methods personally
is the use of an iron IMAC HPLC column for pulling out phosphopeptides. In this case,
again, cells are grown up, peptides are digested and these are applied to an IMAC column on
an HPLC and you get very nice selectivity and specificity of phosphopeptides versus
non-phosphopeptides and this can be used both for direct measurement of the alluded peptides
or for fractionation for further downstream analysis. The authors found that they had
very good selectivity of the methodology of mostly phosphopeptides and non-phosphopeptides
and they also found that there was not a difference between mono and multiple-phosphorylated peptides.
Finally, the last messages to be aware of is there are companies such as Agilent that
make robotic apparatus for doing all of these liquid handling steps for the phospho enrichment.
These systems can have either iron or titanium cartridges for automated phosphopeptide enrichment.
In this case, there's the same basic steps where the resin is there, the peptides bind,
they get washed and then the peptides are alluded off. The company reports greater than
99% of the total phosphopeptide signal is concentrated in a very small volume and the
benefit of a method of this is that it's all robotically done and doesn't have to be done
by hand. In an independent evaluation, the authors found that the iron IMAC worked very
similarly to traditional titanium pulldown methods, very similar identification of phosphopeptides
and different replicates and across different runs, similar quantitational results as well
but much less time involved in the robotics versus the manual handling. Interestingly,
as you can see in panel Panel E, principle component analysis of the 3 methods identified
very different subsets. The 3 methods could be very easily separated indicating that again
they identify non-overlapping subsets and potentially each could be used with complementary
results. 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 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. 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
spectometrist. 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.