Mass Spectrometry

Question 1

What is mass spectrometry?

Answer 1

Mass Spectrometry (MS) is a (bio)analytical technique used to determine the molecular mass of a sample, or of its parts 
Used in biology and medicine mainly (protein structure and proteomics)
e.g. crime scene, detecting drugs and molecular interactions to determine drugs

Question 2

Describe isotopes?

Answer 2

Atoms of the same chemical element have the same number of protons but differ in number of neutrons

Carbon/Hydrogen are the most common elements in biomolecules
Both have more than one stable isotope
C-12 (99%) and C-13 (1%)
H-1 (99.98%) and H-2 (0.02%)
For every 100 carbon atoms, 99 will be carbon-12 and 1 will be carbon-13

Question 3

How does the mass spectrometer work?

Answer 3

MS measures mass to charge ratio
It is a gas phase ion technique
The ions are analysed in vacuum

Question 4

What are the stages of mass spectrometry?

Answer 4

Vaporisation/Ionisation 
Acceleration 
Ion Drift
Detection 
Data Analysis 

Ionisation - Matrix assisted laser desorption/ionisation (MALDI) and Electrospray ionisation (ESI)
Analyser - Quadrupole, Time of flight (TOF) and Orbitrap
Detector - Multichannel plate and Induced current

Question 5

Give an overview of ionisation?

Answer 5

Transfer analyte (solid/liquid/solution) into the gas phase as an ion
It can have positive or negative polarity
Proteins are normally positive but nucleotides makes sense to be negative
Single or multiple charge on molecule (m/z)

Types: electron impact, electrospray and matrix assisted laser desoption ionisation

Question 6

How does electron impact work?

Answer 6

It is the traditional method for producing ions in MS
It involves bombardment of the sample with a high energy beam of electrons
When the electrons hit the sample they sometimes remove another electron

The electrons fired are not absorbed
It can often result in fragmentation

M + e- ——> M+ + 2e-

Question 7

How does ionisation by Electrospray work?

Answer 7

Sample is dissolved in a volatile solvent
Forced into a fine hollow needle
This is connected to the positive terminal of a high voltage supply
The mixture is sprayed as a fine mist of droplets with the solvent evaporated
The sample picks up a proton (H+ ion) or sometimes ammonium salts are used

M + H+ ——> MH+

‘soft’ ionisation technique as it is less likely to fragment the sample

Question 8

What is matrix assisted laser desorption ionisation (MALDI)

Answer 8

Analyte is co-crystallised (dried up) with matrix and irradiated with UV laser
Heats up the sample - almost like heat shock

Matrix - organic chemical compound
This means the sample won’t get destroyed as it is imbedded within the matrix

Solid to gas phase transfer
Primarily single charged molecules

Question 9

What are the mass analysers?

Answer 9

Separate ions according to their m/z
Ions can be manipulated in magnetic fields or electric potentials

Quadrupole – motion in an oscillating electric field
Time of Flight – velocity of ions after acceleration
Orbitrap – frequency of oscillation along electrode

Question 10

Describe the quadrupole?

Answer 10

Four rods arranged symmetrically around beam axis
Opposing pairs are electrically connected
The ion moves in a wavy motion, moving to different rods
It is a filter as to large can crash but too small can escape
With fixed U and V only one m/z can pass
Scanning these voltages transmits other ions

Question 11

What are the advantages and disadvantages of the quadrupole mass analyser?

Answer 11

Cheap & simple
Low mass resolution
Often used as ion guides (transmission only without m/z selection)

Question 12

What happens during time of flight?

Answer 12

They ions are separated according to how fast they travel through a ‘drift chamber’
They have different velocities because of their different masses

The ions are accelerated with the same kinetic energy because of the electric field they are in
Atoms with a lower mass have a higher velocity so arrive at the detector before those with a higher mass

Question 13

What are the advantages and disadvantages of time of flight?

Answer 13

Almost unlimited mass range
Fast
Requires ion packets (pulses) e.g. MALDI

Question 14

What is an alternative time of flight mass analyser?

Answer 14

Orthogonal acceleration TOF
Ions are accelerated but move through the flight tube orthogonal (perpendicular) to the initial direction
Pulsed extraction improves resolution

Question 15

Describe the orbitrap mass spectrometer?

Answer 15

Ions are trapped in a cell and oscillate along a central spindle electrode
Frequency of oscillation is proportional to the ion’s m/z

Question 16

What are the advantages and disadvantages of using orbitrap mass analyser?

Answer 16

Slower scanning than TOF or quad
Much higher resolution
Very good mass accuracy
But expensive

Question 17

Give a comparison of the mass analysers?

Answer 17

Choice of analyser will depend on application

Quadrupole – often present in hybrid instruments, great ion guide and ‘mass filter’

TOF – orthogonal TOF particularly versatile, small – very large molecules

Orbitrap – proteomics – large scale protein ID and quantitation

Question 18

What are some considerations for instrument choice?

Answer 18

Mass range
Resolution – get distinct signals from close m/z
Mass accuracy
Sensitivity – signal response per unit of analyte
Speed

Question 19

What are some factors we need to remember when analysing mass spectrometry graphs?

Answer 19

Electrospray MS show multiple ‘charge states’ for a 10 kDa protein
They are still the same protein but due to different charges we have different signals

At higher masses, we are less likely to observe monoisotopic peak (this will depend on resolution)
Due to this protein masses are usually quoted as average mass

Question 20

How would we find the primary stucture of a protein?

Answer 20

Use:
Chromatography
Intact protein mass
Protein digests
Tandem MS (MS/MS)
Post-translational modifications

For chromatography:
Gas chromatography
Liquid chromatography: size exclusion, affinity, ion exchange, normal phase, reversed phase… etc

Question 21

Describe RP-LC ESI-MS?

Answer 21

Reverse phase-liquid chromatography electrospray ionisation mass spectrometry

Reversed phase: Stationary phase more hydrophobic than mobile phase
We use reversed as the proteins are amphiphilic
Proteins stick to the hydrophobic column and eventually comes off due to salt/buffers
From a spectrum we can use - deconvoluted data (to find the average masses - around the isotopes)

Question 22

Give an example of how PTM can help with determining the primary structure?

Answer 22

Kinetics of phosphorylation

There is a peak but then adding ATP will increase the mass but decrease in intensity
There will be a secondary peak at a low intensity as it is a real time experiment - this can be due to post-translational modifications

Question 23

How can we identify a protein from a sequence?

Answer 23

Identify a protein (and PTMs) from sequence - whole proteins difficult to fragment
Digest the protein
Make peptides using protease (e.g. trypsin)
Peptide separation with RP-LC and ESI-MS
Sequence each peptide in turn (MS/MS)

Question 24

How does trypsin work?

Answer 24

Cleaves C-terminal to K and R residues
K and R are basic residues
Sequester ionising proton
Promote certain fragmentation

Frequency of K and R
Average peptide length 15-20 residues
Ideal for CID fragmentation
Good retention for LC-MS separation and sequencing

Example: Cytochrome C - tryptic digest
Has different heights of peaks - different intensities aren’t very meaningful

Question 25

Describe tandem MS?

Answer 25

Mass analyser 1 : select m/z for fragmentation
Mass analyser 2 : determine m/z of the fragment ions
Triple quadrupole MS, Q-TOF and ion trap - orbitrap

Question 26

Describe the types peptide fragmentation?

Answer 26

Collision induced dissociation (CID)
Collisions with a background gas raise the internal energy of ions and induce fragmentation
It is very clean - will only break in one place at the weakest bond

Radical induced (electron capture/transfer) ECD, ETD

Radiation induced (blackbody, infrared or UV) BIRD, IRMPD, UVPD

We cannot distinguish between isoleucine and leucine by mass as they are just arranged differently

Question 27

How can we identify high order structures?

Answer 27

Native MS
ESI charge states
Xlink-MS
HDX-MS
Covalent labelling

MS - looks for change in mass
MS/MS - determines location of label

Question 28

Describe native MS?

Answer 28

Doesn’t denature the protein - uses aqueous solution and volatile buffer (not acidic or organic)
Look at: non-covalent interactions, ligand binding, stereochemistry
Produces fewer peaks
Folded proteins have lower charge states (higher m/z)
Fewer surface accessible side chains
If denatured - enhances ionisation by increasing sites available for protonation

Example: phage assembly
We can see interactions between subunits

Question 29

Describe Xlink-MS?

Answer 29

Chemical crosslinker (e.g. K or C reactive) with defined length
Bridges two amino acid side chains if they are in proximity
Also “open ended” or intrapeptide crosslinks occur
Chemical crosslinks show proximity of two groups or domains, intra or inter-protein
Allows modelling of low-resolution structures, often in combination with EM

Question 30

Describe HDX-MS?

Answer 30

Protein exchanges some protons (amide backbone) in D2O
Experiments are done by comparing 2 states (2 diff. conditions, e.g. +/- ligand or binding partner) - which exchange the fastest

1. H/D exchange (any temperature) - exchanging outer surface hydrogens to deuterium
   The others are ‘protected’ and can’t be exchanged
2. Quench (low temp and pH 2.5)
3. Proteolysis
4. Cooled LC-MS
5. Data Analysis

Question 31

Describe covalent labelling?

Answer 31

Protein Footprinting
Probe solvent accessibility by labelling side chains
Fast Photochemical Oxidation of Proteins (FPOP)

Irreversible, covalent modification of surface-exposed amino acid side chains by hydroxyl radicals (H2O2 +UV laser)
Digest & peptide sequencing
Oxidation increases mass by 16 Da
Use MS to determine oxidation sites
Footprinting - FPOP, Hydrogen-deuterium exchange, chemical cross linking

Question 32

What are some application of mass spectrometry?

Answer 32

Antibody characterisation
Biopharmaceuticals
Virus assembly
Amyloid aggregation
Assembly of complexes
Protein unfolding