Mass spectrometry

Question 1

Overview of mass spec

Answer 1

Get ions from liquid or solid phase into gas phase

Can also only measure charged molecules so have to have a way to generate charge (this happens at the source)

Dotted lines because sometimes source is at atmosphere and sometimes at vacuum

Typically mass spectrometer is at vacuum

Sperate ions by mass to charge ratio (in analyser) - under high vacuum

Then detector to detect ions

Then need an acquisition system to bring all the data together

Question 2

Methods of sample introduction

Answer 2

Gas chromatography (volatile ions) - not going to focus on this

Syringe pump - fill syringe with protein and solvent and pump into mass spec

Liquid chromatography - level of operation to reduce complexity of sample before it enters mass spec

Capillary electrophoresis - take capillary and fill it with a buffer e.g. phosphoric acid along with protein of interest, apply an electric field and the protein will migrate within the capillary

Solids - going from Solid phase into gas phase

Question 3

Methods of ionisation

Answer 3

Electron ionisation

Chemical ionisation

Fast Atom/Ion Bombardment

Secondary Ion Mass Spectrometry

Field desorption

Plasma desorption

Electrospray Ionisation

Matrix Assisted Laser Desorption Ionisation

Electrospray Ionisation and Matrix Assisted Laser Desorption Ionisation are the two most important ionisation methods for biological mass spec due to being soft ionisation techniques (don’t fragment molecule and so retail the molecular ion)

Question 4

Primary technique now used for electroscopy ionisation

Answer 4

Go from liquid phase to gas phase ions

Metal capillary containing a liquid

Apply a high potential (around 3000 V) to get an electrophoretic effect and forms a Taylor cone

A spray of charged droplets forms

These undergo desalvation - basically evapiratio. Where droplets get smaller and smaller until gas ions are left to put into the spectrometer

Question 5

Basic principles of MALDI ionisation

Answer 5

Take molecule of interest mix with a matrix (low molecular weight absorbing compound)

Spot this onto target plate

And dry it onto surface

Now have sample embedded into a crystalline matrix e.g. α-cyano-4-hydroxycinnamic acid, Sinapinic acid, 2-amino benzoic acid

The matrix absorbs UV light in a certain wavelength

So use laser at this wavelength, matrix flakes/comes off surface and part of the energy is imparted onto the sample and hence that becomes ionises and comes off the surface and becomes charged

Question 6

Once ions are generated, what is next?

Answer 6

Need a way to separate them - Use time of flight (ToF) mass spectrometry

Question 7

Describe how the analyser - Time of flight (ToF)

Answer 7

In vacuum - measure the time it takes for the ion to travel to the detector

1. Fire UV laser at sample, generate ions
2. Start timing
3. Stop when they reach detector

The lighter the ions the faster they move

Equations are worked through to give value that is m/z

Question 8

Describe the analyser - Quadrapole analyser

Answer 8

Mass filter – separation accomplished using combination of DC and RF electric fields

Ions oscillate through filter towards detector

Only ions with stable trajectory transmitted

By scanning DC/RF ⇒ m/z

4 metal rods, there’s an AC potential and a DC potential

Can change the DC voltage to make one side more or less positive/negative

Can change this to let particular m/z values through - it is a mass filter

Question 9

Last generation of analyser - Q-TOF instrument

Answer 9

Quadrapole TOF : Hybrid of the two analysers discussed previously

ion mobility separator - used in structural biology - slow down ions, operate by surface area by filling area with helium and passing ions through - adds another dimension of separation

Time of flight analyser uses W optics, means the flight time is slower so more time for separation, therefore higher resolution that linear ToF analyser