Learn about the technologies underlying experimentation used in systems biology, with particular focus on RNA sequencing, mass spec-based proteomics, flow/mass cytometry and live-cell imaging. A key driver of the systems biology field is the technology allowing us to delve deeper and wider into how cells respond to experimental perturbations. This in turns allows us to build more detailed quantitative models of cellular function, which can give important insight into applications ranging from biotechnology to human disease. This course gives a broad overview of a variety of current experimental techniques used in modern systems biology, with focus on obtaining the quantitative data needed for computational modeling purposes in downstream analyses. We dive deeply into four technologies in particular, mRNA sequencing, mass spectrometry-based proteomics, flow/mass cytometry, and live-cell imaging. These techniques are often used in systems biology and range from genome-wide coverage to single molecule coverage, millions of cells to single cells, and single time points to frequently sampled time courses. We present not only the theoretical background upon which these technologies work, but also enter real wet lab environments to provide instruction on how these techniques are performed in practice, and how resultant data are analyzed for quality and content.
Lecture 1 - Basics of Mass Spectrometry 1
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Do curso por Icahn School of Medicine at Mount Sinai
Experimental Methods in Systems Biology
150 classificações
Icahn School of Medicine at Mount Sinai
150 classificações
Curso 2 de 6 em Specialization Systems Biology and Biotechnology
Na lição
Mass Spectrometry-Based Proteomics
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Marc Birtwistle, PhDAssistant Professor
Department of Pharmacology and Systems Therapeutics, Systems Biology Center New York (SBCNY)
Hello everybody, welcome to week three of the coursera course and
experimental methods in systems biology.
Today we're going to be going over mass spectrometry based proteomics.
And first, just as last week I'll go over a few lectures
with slides in terms of the theory of, of mass spectrometry for proteomics.
And then, we'll actually go in the lab and
see how some of these techniques look in practice.
So, for today, I'll be going over a few things and
break it into a few lectures for you.
Just as I've done last week.
First, we'll just explain a little bit.
What exactly is mess, mass spectrometry and, and
how can it be used to quantify and identify proteins?
second, we'll go into how does a mass spectrometer work.
What are all the different components and what are the techniques and, and
the pieces of separate equipment that are,
that work together in order to comprised a mass spectrometer?
And lastly, we'll devote quite a bit of
time to quantitating mass spectrometry data.
And, what are the different techniques out there?
What are their pros and cons and how are they new?
So first, what exactly is a mass spectrometer?
Well, it's simple an instrument where one can have ions,
which are charged atoms or charged molecules.
And then, you can separate these ions according to their mass-to-charge ratio.
So, what is their atomic weight,and what is their charge?
Divide one by the other.
And then, based on the separation, one can then quantify the number of
ions as a function of their mass-to-charge ratio.
Now, there are two main ways in which mass spectrometry is used for
analysis of proteins or protein identification.
One is called peptide mass fingerprinting, which is used less and less nowadays.
But, it's still something that is, that has been routinely used and
is used from time to time.
so, I'll just briefly mention it here, but not go into it in much detail.
But, it's simply if you have a protein that you're interested in,
usually a relatively pure protein you can proteolytically digest
that with for example trypsin or chimotrypsin or another.
Pretty ace with a, a specific cutting pattern.
You digest it into a series of peptides and then look
at the mass-to-charge ratios of all of those peptides which you've digested.
One of the benefits of this is that it's quick.
And you can get a so-called fingerprint of the digestion
of your protein into different weights of peptides very easily.
But, the drawback is that it's not really able to handle mixtures.
And, and, most of the time, you have a sample from a tissue or
cells where you have a whole mixture of proteins.
And you'd like to identify what sorts
of different proteins are changing across the samples.
So, for those purposes which is the bulk of what people call proteomics
with mass spectrometry experiments.
Nowadays, this tandem mass spectrometry also called MS/MS.
And this is based, again, on, usually on,
on proteins breaking do, broken down into peptides for your proteolytic digestion.
But when you run experiments with two mass specs and, and series,
you can do many more things than you can with the peptide mass fingerprinting.
First of all, you can have a much greater ability to deal with, printing mixtures.
You can also, have pretty good confident,
confidence in analyzeing the sequence of the peptides, and also identifying and
quantifying post-translational modifications on the peptides.
Of course you know,
the, these tandem mass spec experiments can be quite expensive and time-consuming.
So, that's a trade-off, but most of the time the information that
you gain from doing tandem mass spec is, is well worth the higher cost.
Okay. So,
what exactly is tandem mass spectrometry?
Well, it's, as I said,
when you have two mass spec, spectrometers arranged in series.
And, in the first mess, mass spec the ions are separating
separated according to their mass to charge ratio, m to z.
value, in the first stage mass analyzer.
And then, based on this you select certain ions with
a very particular m over z value for further analysis.
These peptides are then broken into fragments and these fragments
are analyzed in a second mass analyzer they in the second mass spec stage.
so, based on this fragmentation pattern you can figure out protein sy-sequence and
also the types of post-translational modifications that were on peptides.
And, as a pictorial represation, representation of how this might work you
can imagine that, and you have a protein that has been broken down into peptides.
These peptides are made of individual amino acids.
And, when you break these peptides apart by fragmentation into different pieces.
Based on the mass that's missing form each of
those pieces when you go from larger pieces to subsequently smaller, and
smaller pieces you can tell by what mass is missing what,
what amino acid has been broken off from that peptide.
Which then, gives you information about the sequence of that particular peptide.
So, and not only can you tell, get information about the peptide sequence.
Of course, because post translational modifications have mass,
you can tell a lot about the.
Them, so post-translational modifications, of course,
play a large role all throughout many aspects of molecular biology.
A good example here is the multiple modifications that can be
present on histones, which play a big role in gene, in epigenetic gene regulation.
For example, you can have methyl groups,
acetyl groups, phosphate groups on different residues of the histones.
All of which play quite an important role in in regulation of gene expression.
And, all of which can be detected by mass spectrometry because it's such a general
technique that allows you to analyze the mass of molecules which are input to it.
Similarly, signal transduction, is to a large extent regulated by post
translational modifications in proteins where you have signaling reactions that
put phosphates, or acetyl groups, sumoylation groups, ubiquitin groups,
or methyl groups on to various proteins in different amino acids residues where.
When you have these proteins and peptides and input them to the mass spectrometer,
you can see these post-translational modifications by their fragmentation and
detection of their mass.
So just to summarize a little bit,
what you can do with mass spectrometry based proteomic proteomics.
Cardio mix is you know, many things first of all
maybe the simplest thing you can quantify and or compare between two or
more conditions total abundance of proteins in samples.
Based on those total protein abundances.
You can also look at their, their levels of post translational modifications.
You can identify new post translational modifications and or
you can detect the levels of them across different samples.
You can look at Phosphorylation.
You can look at Acetylation, Methylation, Methylation,
hydroxylation, glycosylation and, and many, many more.
Essentially, if the modification that you care about has mass,
and it can be ionized and then fragmented in a, into a particular pattern,
from which you can deduce which residue that it was attached to.
Then, it can almost certainly be analyzed by mass spectrometry.
So, how does tandem mass spectrometry actually, work?
What are the steps that go into it?
So, of course, first of all, you have your sample which you've
isolated from a tissue, or cells or some other source.
And, you isolate your proteins from it.
And, because there are usually many, many different types of proteins, and a sample.
And, these proteins can stand quite a large dynamic range from you know, on
the levels of micromolar sometimes all the way down to the levels of manimolar and
picamolar sometimes.
It's important to separate and
enrich the proteins in your sample and the peptides in your sample.
This allows you to reduce the complexity of your mixture.
And, it allows you to get a much deeper analysis of
what's going on in your sample.
So, with this separation and enrichment, almost always then,
the whole proteins are digested into peptides.
There is a branch of proteomics, called top-down proteomics,
which attempts to analyze proteins without their proteolytic digestion into peptides.
But this is a relatively small, aspect of the proteomics field.
And, mostly the proteolytic digestion is used to turn your proteins into peptides,
which is not too dissimilar from RNA sequencing where we take our,
entire transcriptome and then fragment them into small pieces.
then, these peptides in order to work in mass spectrometry they have to be ions.
They have to have a mass to charge ratio.
So, there is a step of ionization where you take your peptides, and
you ionize them so that they can then be analyzed for
their mass to charge ratio in the first mass analyzer.
And typically this first ma, mass analyzer is used in a, kind of a scan and
select mode, where're looking for, what are the most abundant peptides that
happen to be coming into the mass spec at this moment.
And, let's select those and send them into a fragmentation chamber,
where we can take those peptides.
You then, break them into smaller pieces and send them into the second mass
analyzer, so that we can gain information about what is the sequence of
that peptide that happens to be coming through the mass spec at this moment.
And, also, so,
we can get more information about the amount of that peptide coming through.
and, at the end of this process what we get are.
So, called mass spectrum which plot on the y axis intensity of a particular
ion coming through versus the actual mass to charge ratio of it.
So, what do the data that come off of one of these mass spec runs look like?
So, here's just a small example of.
They tend to mass spec data from a tandem mass spec experiment,
where you can imagine we have this mixture of peptides here shown by
the cartoon of different colors, color ribbons here.
And, as they pass through the, the, as they're ionized, and
then they pass through the first mass analyzer, you can see peaks.
That particular mass to charge ratios here and these peaks have a certain intensity.
As I noted, the intensity is on the y axis here and then the peaks
of different mass to charge are, are shown here on the x axis.
And, you can see there's a peak here with a mass to charge ratio of 568.65,
and two more here.
One of them here at 836.47 is currently being selected for spec analysis.
So, first mass analyzer isolates this mass-to-charge ratio and
sends that on for fragmentation and second, a second mass spec analysis.
So, this peptide gets fragmented into various pieces as you
can see here where again mass to charge ratio
over the intensity of each of these different mass charge ratios.
So, as you can see there's just a series of here.
And, by looking at the differences and these peaks,
we can start to put together what are the amino acids that are,
when removed from each of these peaks give rise to the mass that we're missing.
And, that allows you then to deduce the sequence of the peptide,
which is shown here in the upper left corner.
And and, this is typically done by software.
and, and it's a totally automated process but
you can also do it by hand by looking at differences.
For example, you can see some of the differences here between peaks,
a difference of 57 here between these two peaks.
That implies a glycine difference of 163.
Here between these two peaks implies a tyrosine,
difference here of 99 implies a valine, and so on and so forth.
You can also detect different post translational modification states
as I noted before here.
If you have a, a cysteine down to a nitric oxic,
nitric oxide modification, it gives rise to a certain mass fingerprint.
So, this kind of gives you an idea of,
the type of data that a mass spec gives you, and then.
How do you actually use that information to determine both
the quantity of peptides that are, that were present in your sample?
But, also what's the sequence of those peptides and
potential post-translational modifications on each residue within those peptides.
So, in the next series of lectures, we'll go into a little bit more detail on
how do each, how does each piece of this mass spec actually work.
Like, what are the, what's the engineering design, and what are the principles by
which you can actually analyze And do these sorts of things.
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