Lecture 2 - Basics of Mass Spectrometry 2

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En provenance du cours de Icahn School of Medicine at Mount Sinai

Experimental Methods in Systems Biology

142 notes

Icahn School of Medicine at Mount Sinai

Experimental Methods in Systems Biology

142 notes

Cours 2 sur 6 dans Specialization Systems Biology and Biotechnology

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.

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Mass Spectrometry-Based Proteomics

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Lecture 1 - Basics of Mass Spectrometry 115:38

Lecture 2 - Basics of Mass Spectrometry 215:03

Rencontrer les enseignants

Marc Birtwistle, PhD
Assistant Professor
Department of Pharmacology and Systems Therapeutics, Systems Biology Center New York (SBCNY)

So, last lecture was just a very brief introduction to mass spectrometry base

proteomics and explaining to you a little bit about what it can measure.

This lecture we're going to go into more of the technical details of

how Tandem Mass Spectrometry actually works and

what, what are the different components that make up a Mass Spectrometer.

So, if you remember we went over this general work flow for

a Tandem Mass Spectrometry set up last lecture.

So, we'll just review it briefly here.

You're starting with a sample.

Either from a tissue or, cell culture, where you've,

isolated proteins and typically one separates and

enriches, those proteins for, based on the types of things one is looking for.

And this separation and enrichment can actually be quite extensive.

One very common way to separate proteins is just like

as an elestrin blot to separate by molecular weight and

then analyze different bands of molecular weight to reduce complexity of the sample.

Almost universally proteins in a sample are digested

to turn the proteins into peptides, a common protease you

use to do that is trypsin but there are other proteases which are sometimes used.

And so then you go from a usually fairly highly complex mixture

of proteins to a simplified and a more simple mixture of peptides.

These peptides are then typically separated even further.

And then input into the mass spec via an ionization process.

After they're anano, ana, ionized.

They can actually be analyzed via mass spectrometry based on

their mass-to-charge ratio and input into a first mass analyzer,

which is used to, as a separation device to pick out which are the most

abundant peptides that are being input to the mass spectrometer at this stage.

And then select those ones for

further fragmentation sent into a second mass analyzer which analyzes the fragments

of those peptides, from which one can deduce protein sequence and

also post-translational modifications and relate that to the, amount or

quantity of the peptide that was coming in to the mass spectrometer.

So, one of the most common forms of

separation in mass spectrometry is liquid chromatography.

in, in many cases a liquid chromatography column is the step which is,

interfacing a mixture of peptides.

And the mass spectrometer it, itself through a ionization process we'll

describe a little bit later called electro-spray ionization.

So liquid chromatography setup usually consists of many pumps which is

taking your sample usually diluting it with some kind of solvent.

This solvent composition can change over time, which changes its affinity for

the most important part of the chromatography setup, which is the column.

The column is packed with a resin.

A common resin for proteomics is a hydrophobic C18 column but

there are many types of columns.

Which can separate your mixture of peptides based on

various physical chemical properties.

Such as hydrophobicity or charge for example.

And the affinity of peptides for

this column can be changed by changing the solvent composition.

So typically you would, you inject your sample onto this chromatography setup,

it starts to be pumped through the column,

and as time goes on you can change the solvent composition on

a so-called gradient which then changes how your peptides elude from this column.

And then by analyzing what comes out of the end of this column over time we

simplify a mixture of peptides into an even, a simpler and purer, composition

of peptides coming out and being input to the mass spec at any given moment.

When we input our sample of peptides to the liquid chromatography column we

have to have a way of inputting that into the first mass analyzer.

And to ionize the peptides that are coming off of this column.

The method that's used to do this is called electrospray ionization.

There are,

of course, many types of ionization that some of which are listed here.

But the two of which which are commonly used in proteomics

are the electrospray ionization and so called MALDI or matrix-assisted laser

desorption ionization which I'll explain in the next two slides.

So electrospray ionization was one of the key breakthroughs which allowed

proteomics to really take off in,

in mass spectrometry applications where one is able to interface

the output of a liquid chromatography column to a mass spectrometer.

And the idea is, actually remarkably simple,

although technically very challenging, is to take that flow output from

the liquid chromatography column and put it out of a very fine spray needle,

which turns the the liquid coming out into droplets, and

those droplets are, are subsequently dried, and then,

with a high voltage being applied between the liquid chromatography column and

the input to the mass spectrometer these, these peptides can become ionized and

then can be input into the mass spectrometer.

And this invention, the Electrospray Ionization technique,

was actually awarded the 2002 Nobel Prize, because it allowed not only mass

spectrometry based proteomics to take off but many other forms of mass spectrometry,

which depends on separation by a liquid chromatography column.

Another commonly used form of ionization in

mass spectrometry based proteomics is called MALDI.

Where you simply embed your peptides in a so called matrix.

Which is able to transfer charge to

your peptides when you shine a laser on the sample.

So you put your sample on a so called target, and shine the laser on it,

and there's a potential then between the target and the mass analyzer.

So a, a laser is shined onto your sample some charges are transferred

over to your peptides they become ions and because of the applied voltage

they then travel into the mass analyzer where they can be further analyzed.

So after your peptides are ionized, they can be analyzed by a mass spectrometer.

And the, the functional unit of the mass spectrometer that

analyzes things based on a mass to charge ratio is called a mass analyzer.

So there's, of course, several types of mass analyzers.

Three commonly used ones in proteomics are a quadrupole,

a time of flight analyzer, and an ion trap, or an orbitrap analyzer.

So a quadrupole analyzer works in the following way according to this schematic,

where the peptides, or ions, are input into

a source from the electrospray ionization source, for example.

And then they travel through a column where you have four

rods two of which are positively charged and two of which are negatively charged.

So that there's voltages across these rods and

then the voltages are changed in a time dependent manner.

So that you can imagine if you're an ion traveling through this series of columns,

that you oscillate then either towards the positive or

towards the negative nodes in this, in this quadrupole.

And based on the time-dependent behavior that's, that's induced on these voltages,

which can be tuned, you only allow certain ions with,

with a particular window of mass to charge ratios through the other end.

To the detector so a quadrupole can act both as a, a filter and

also as a detector for certain mass to charge based on our ability to

change the time dependent behavior of the voltages applied to the quadrupole.

So another type of mass analyzer which is typically interfaced with multi ionization

is the time of flight mass analyzer, where as described before.

You shine a laser onto the, onto your sample on the MALDI target, which then

induces a, a pulse of ions which are then transferred into the mass analyzer.

So. If you can imagine that you

have a fixed voltage at one end of the time of flight

mass analyzer the ones with higher mass to charge ratio travel at

different speeds as the ones with smaller mass to charge ratio.

So because we can have a, a very defined start point for

when the ionization occurs, and then we can measure the times at

which different mass-to-charge ratios arrive at the detector.

On the other end of the time-of-flight mass analyzer,

we have a way of determining how much of each mass-to-charge is in

our sample by analyzing the times of arrival of each time of flight of,

of each ion through the time of flight mass analyzer.

The last and most sophisticated but is becoming much more common in proteomics

in the past few years is a so called Orbitrap which is a type of ion trap.

As you can see here on the left,

it's not a very large device, as big as perhaps a common coin.

but, the way that the Orbitrap works is,

a packet of ions is injected into the Orbitrap, with, with a linear velocity.

And then, there's a voltage that, that's applied to the inner core of

this orbitrap, which then gets the ions to start spinning around the orbitrap.

And after a short burn in period, the ion, the ions reach a stable circular orbit

around this orbitrap, and The unique thing about the Orbitrap is that, because

of it's shape the ions then, although they're spinning around the cylinder,

they also oscillate radially across the axis, of the cylinder.

And the period of that oscillation radially is uniquely

relatable to the mass-to-charge ratio.

So by detecting how these ions are oscillating radially across

the Orbitrap in time.

And then applying some advanced mathematical processing to those signals,

namely Fourier transform-based mathematics.

One can determine what is the mass to charge spectra and intensities

of these mass to charges of the ions that have been injected into this orbitrap.

So I can say there's, there's a lot of good information and

videos about this orbitrap system on the web link I've put here on the right.

So I've talked a little bit about separation, ionization,

the different types of mass analyzers but I haven't talked how that do

you actually couple those mass analyzers together through fragmentation.

And what are some typical combinations of

mass analyzers that one uses in a tandem aspect setup.

So a very common method of fragmentation is so

called fast atom bombardment or collision induced dissociation.

Where essentially you,

you have your ions in a chamber and you simply bombard them with, with atoms.

Which induces their their fragmentation and

so in many cases ions are collided with an inert gas such as helium

which then just causes them to randomly fragment causing the types of

fragmented spectra that we, that I showed you a sample of in the first lecture.

And this is a very common interface between two mass analyzers.

One very common combination is a quadrupole with an orbitrap.

So the quadrupole allows you to filter certain whole

peptides through whichever are the most commonly and most

abundant ones that happen to be coming off of the liquid chromatography column.

And being amp, ionized by the electric spray ionization at the current time.

Which you can then select for

fragmentation via, for example, fast atom bombardment.

And then take those fragmentation products put them into an orbitrap and then

analyze what are the different fragments that I have here that were a result of

fragmentation of this particular peptide that was selected by my quadrupole.

So there's, there's a very nice and

beautiful animation of this from Thermo Fissure who

makes a quadrupole orbitrap tandem mass spect system called a Q Exactive.

a, I can highly recommend you to go to the website here.

I've put on the left and, and check this video out to really see you know,

what happens to these ions as they go through the tandem mass spec process.

And in a few lectures we actually take you into a lab to show you

how does one actually use such a Q Exactive machine and

how does one have to actually prepare a sample so that you can get data.

And, and input your sample into such a Q Exactive machine.

So in the next lecture I'm going to be talking about how

you can take this mass spectrometry data.

And how you can design your experiments in such a way such that you can

quantify the resulting data.

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