>> Hello and welcome to the next in our OSTR Series on cutting edge methodologies. Today
we're going to discuss methods for global discovery experiments and mass spectrometry.
In mass spectrometry, there are generally 2 ways that we go about performing an experiment
to do large global analyses and these are either termed DDA or DIA with DDA representing
data dependent acquisition and DIA, data independent acquisition. As you can see in the figure
here in a data dependent experiment, the presence of a precursor ion triggers its isolation
and fragmentation for analysis. Fragment ions have a direct linkage to the parent peptide
mass. An acquisition is dependent upon the data in the mass spectrometer, hence the name
data dependent. In contrast, in a data independent experiment, mass windows are cyclically isolated
and scanned with all of the ions within the window fragmented simultaneously. The particular
fragment ions for a given peptide have to be extrapolated out of the pool of fragment
ions for all peptides in the window. Acquisition is independent of the data in the mass spectrometer,
hence the name data independent acquisitions. So, if we just look a little further again
what you see in the DDA experiment is that ions come into the mass spectrometer, are
isolated in the quadrupole and then analyzed in a full scan mass spectrum. Based on the
ions detected there, specific ions are selected for fragmentation based on either their intensity
or their charge state and each one is individually isolated and fragmented and then analyzed
again. So again, we have a direct linkage between the parent ion mass and the fragment
ions that come from it. However, there is a limitation in the number of ions that can
be fragmented per parent ion scan. In contrast, in the data independent scan, the ions again
go into the quadrupole, a full mass spectrum is analyzed, and then sequential precursor
windows are isolated and all of the ions that fall within that mass are isolated, fragmented
and a mass spectrum combined. These mass windows tend to be on the order of 4 to 25 m/z units
and so you get a combined picture of everything that's present, nothing is lost, there's no
limit on the number but the linkage between the parent ion mass and the fragment ion mass
is lost, so both methods have advantages and disadvantages. DDA experiments again maintain
the link between the parent mass and the fragment ion spectrum and they require only a sequence
database for peptide and protein identifications. However, due to the stochastic nature of ionization,
some peptides will be missed. Also, missing values replicate to replicate and across sample
will affect the ability to do good quantitation especially across multiple experiments. DIA,
all experiments, all peptides, excuse me, are fragmented and can theoretically be analyzed,
so there are no missing values and there's more complete quantitation in values across
replicates. However, in a single fragmentation window, multiple precursors are fragmented
and the direct link to the parent ion is lost. Therefore, spectral libraries are required
for database searching such that the fragments can be matched to a known library, pulled
out and then continued to be searched. Optimally, these libraries should be collected on the
same instrument and using the same LC conditions as the final run, so in that case, you still
need some DDA experiments for every DIA experiment. Some instrument manufacturers such as SCIEX
have also managed to modify the isolation windows and instead of having static windows,
they offer variable window isolation. In this method, the isolation window is varied such
that it's narrower in the mass ranges where there are a lot of peptides and wider in the
mass range where there are fewer peptides. This means that there's higher specificity
in the isolation, there's better signal to noise and hopefully better data across the
entire experiment. So, within a DDA experiment, there are also different ways that one can
perform quantitation to get relative information about protein abundance. First, our label
free methods such as you see on the top in which cells are grown in normal media, the
peptides, proteins, excuse me, are digested to peptides, the peptides are isolated and
then analyzed by the mass spectrometer. Quantitation is generally performed from the parent ion
mass using an extracted chromatogram which looks a lot like an HPLC profile. There are
also methods such as SILAC in which cells are grown and isotopically labeled media.
The media can, the cells can then be combined and digested and analyzed on the mass spectrometer.
Again, we're doing quantitation of the parent ion masses from the extracted ion chromatogram
but the different states of the cell have a slightly different mass due to the isotopes
and so they can be directly compared quantitatively. There's also a third approach that's commonly
used in which cell proteins are isobarically tagged. In this case, again, peptides are
isolated, they're digested and then they are reacted with a reagent that has an isobaric
tag such that the mass that gets added, regardless of state, is the same. Then when the peptides
are introduced to the mass spectrometer and fragmented, a reporter ion will come off with
a low mass that differs depending on which form of the tag was put on the protein, whereas
you can still see the peptide sequence information. There are generally 2 types of these tags,
either TNT or ITRAQ depending on the manufacture. The ITRAQ tags come in a 4-plex and an 8-plex,
which allows multiple different samples to be combined and analyzed at once. And TNT
tags currently come up to an 11-plex, again, 11 samples could be combined and analyzed
at once. Each of these methods have different advantages and disadvantages just like DDA
versus DIA and the way to proceed often depends upon several questions about sample and method
development. First, what type of sample are you analyzing? Is this a cell line or an animal
model or patient sample? Cell lines are readily amenable for example to the SILAC method.
You can get SILAC-labelled CHOW though it's quite expensive and patient samples really
cannot be done in a SILAC method, so in that case either label-free or TNT ITRAQ type methods
are best. Additionally, whether the sample is a more traditional sample that one wants
to look at or for example microbiome samples. Microbiome, where one is looking at microbes
and host environments, are often done using DIA methods because the identification of
different microbes can be processed over time, the data can go back and be researched as
different microbes of interest come up for analysis. You might also want to consider
how many samples are you analyzing. If there's only a few then a SILAC or a label-free approach
may be good but if there are a lot of samples being analyzed, the benefits of multiplexing
with the isobaric tags come in very handy in terms of instrument and analysis time.
And finally, what type of analysis are you performing? Are you looking at post-translational
modifications or total protein? For total protein, is there are usually multiple peptides
per protein identified, label-free approaches work well. However, for post-transitional
modifications as there's usually only a single peptide with a single modification, some form
of isobaric or SILAC labeling with additional quantitation information often gives better
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 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 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 [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.