Physical Biochemistry Mass Spectrometry 1

A mass spectrometer is an instrument which is used to define the covalent structures of

substances by ionising, separating and detecting molecular and fragment ions according to their
mass to charge rations (m/z).
Mass spec can be carried out with very small samples (as low as 10-18 mole).
It can be used to study very complex molecules such as protein digests.
Purity is not very important.

Ion source → Mass analyser → Detector

1. Generate gaseous ions
2. Charged ions can be controlled (attraction / repulsion)
3. Separate m/z
4. Detection .

Almost all mass spec experiments are carried out under a high vacuum, to prevent ions colliding
with a gas molecule, as this would annihilate the ion.

The mass spectrum is a record of the ions that are detected, intensity (y) Vs. m/z ratio (x)
Intensity does not have units. The most abundant peak is given the arbitrary value of 100%; all
other peaks are plotted relative to this. Always label each axis.

Ionisation:

Hard techniques have a lot of energy input, resulting in fragmentation

of sample.

I) Electron Impact (EI)

§ Hard
§ Ionisation of molecules already in the gaseous phase
§ Small molecules (<1000 Da) – larger molecules are harder to
get into gaseous phase
§ Not a very efficient process (1/1000 will ionise)

1. Sample is introduced into source by heating it from a probe tip until it evaporates, or from an
on-line gas chromatograph.

2. Gas phase sample is bombarded with high-energy electrons coming from tungsten filament
§ A magnet keeps the electrons in a beam, this increases ionisation efficiency

[Page 1]
Physical Biochemistry Mass Spectrometry 1

3. Ionisation occurs by loss of an electron to give M+ (single charged radical cation)

§ There is no collision of electrons (they are very small)
§ An electron from the electron beam comes close to an outer orbital electron, generating
repulsion; the energy is sufficient to expel an outer orbital electron.
§ Losing an electron generates a single positively charged ion.
§ As the species generated has an unpaired electron, it is known as a radical.

4. Most molecular ions decompose into fragments via uni-molecular reactions

Energy = 70eV: average bond energy is 5eV. This excess of energy will not only cause loss of an
electron, but also fragmentation of the cation.

5. The repeller is a metal plate which has a positive charge, it will repel the positive molecular
ions.

M + e- → M.+ + 2e-

II) Fast Atom Bombardment (FAB)

§ Soft / Hard (some fragmentation, but a lot of intact molecules as well)
§ Peptides, oligosaccharides (<6000 Da)
§ Ionisation from the liquid phase (sample is dissolved in a solvent)

1. Liquid matrix and sample mix are put on the end of a probe
§ Low volatility and relatively unreactive solvents are used e.g. glycerol
2. Sample is bombarded with a beam of high energy atoms (inert gas - Xe) / ions (Cs+)
3. The beam strikes the sample, and energy is transferred, the outermost sample / matrix layer
become ionised.
§ Both positively and negatively charged ions are formed (only one type can be analysed at
a time – Use an appropriate repeller.
4. The matrix allows ionisation of a layer at a time, so the experiment can be run over several
minutes. This increases the sensitivity of detection.

Referred to as a ʻdirtyʼ ionisation technique as the matrix is

ionised as well as the sample, and the matrix is often in
excess.
FAB spectrum will contain matrix peaks.
Very sensitive to contamination, especially detergents- the
detergent will always be at the surface of the matrix, so will
be preferentially ionised.

[Page 2]
Physical Biochemistry Mass Spectrometry 1

III) Matrix Assisted Laser Desorption Ionisation (MALDI)

§ Peptides, proteins, DNA (<500,000 Da)
§ Ionisation from the solid phase
§ Both + and – charged ions are produced – only one can be analysed at a time

1. Sample is embedded in a low MW, UV absorbing ʻcrystallineʼ matrix

§ The matrix has a maximum absorbance near to that of the laser being used for ionisation
§ The matrix materials have conjugated ring systems, double / triple bonds to absorb UV
§ α-CHCA is used for peptides and proteins
§ 2,5-DHB is used for carbohydrates
§ UV-Nitrogen laser is often used (wavelength of 337nm)
2. Sample + matrix are added to the end of a metal target / probe
3. Matrix and sample are allowed to dry and crystallise
§ A 3D crystal lattice forms that has the sample embedded within it
4. The matrix absorbs the laser pulse and energy is transferred into the sample to ionise it

“Delayed extraction” has allowed modern MALDI instruments to give well resolved spectra.
§ The pulse of ions is kept in the source for a short time after the laser pulse in order to allow
time for ions formed deep within the matrix to ʻcatch upʼ with those formed near to the
surface.
§ This is carried out by having a time delay before the large potential is applied to repel the
ions
§ Before this there would be more dispersion of ions, reducing the quality of the spectra

[Page 3]
Physical Biochemistry Mass Spectrometry 1

IVa) Electrospray Ionisation (ES/ESI)

§ Soft
§ Peptides, proteins, oligosaccharides (500,000+ Da)
§ Ionisation from liquid phase
§ Occurs at atmospheric pressure
§ Produces multiply charged positive and negative ions

1. Sample is introduced into a thin capillary (glass)

§ The end is coated with a thin metal film (gold)
§ The metal coating allows the generation of a large potential between the end of the needle
and the electrode (~4kV). This will affect the charge of the ions to some extent.
2. The sample (in solution) leaves the needle and comes into contact with a drying gas (N2)
§ The droplet contains solvent and sample
§ The sample will have a net charge based on its pKas (net + or -)
3. The solvent beings to evaporate, the droplet gets smaller; ions are forced close together.
§ The ions repel one another, when the solvent level drops to a certain level the droplet can
no longer contain all of the charged ions – the droplet then explodes into smaller droplets
§ This process then occurs again, until there is no solvent left, just ʻnakedʼ gas phase ions
4. A potential is set in the source in order to send the positive or negative ions into the mass
spectrometer
Early ES machines used solvent systems that delivered μL/min

[Page 4]
Physical Biochemistry Mass Spectrometry 1

IVb) Nano-Electrospray (Nanospray)

§ Operate at flow rates of ~30nL/min
§ More sensitive than regular ES
§ 1μL sample is loaded into needle – this allows for many MS experiments to be carried out
on a single sample
§ Sample quantities are often <1ng
§ Sample can also be introduced using on-line nanoLC (Liquid Chromatography) – a very
powerful method for analysing complex mixtures
§ Analysing molecules in solution is useful, as most biochemical reactions occur in solution

Most modern ES sources do

not spray the sample directly
into the mass spectrometer,
instead they use a Z-spray.

Ions are first deflected into the

cone, then deflected again into
the MS machine.

Neutral molecules (solvent) will

not be deflected, so this
method is a way of increasing
selectivity for the ions.
Increases the MS performance
and sensitivity.

Unlike most ionisation

techniques, ES produces
multiply charged ions. ES
spectra do not contain single
peaks, they contain a series
of different peaks, due to the
different charges. Computer
software can be used to
obtain the correct mass.

[Page 5]
Physical Biochemistry Mass Spectrometry 1

Separation of Ions based on their m/z:

Carried out by mass analysers
Important criteria:
§ Upper mass limit – what is the largest m/z that the analyser can separate
§ Ion transmission – how many ions that are produced can the analyser separate at a time
§ Resolution – How good is the separation of peaks with a very similar m/z

I) Magnetic Sector
Was initially used for isotopic analysis – used to separate U-235 from U-238 to obtain material for
the first atomic bomb.
A magnetic field deflects the different isotopes to varying degrees resulting in separation

m/z = B2R2/2V
B = magnetic field (variable)
R = radius of magnet (constant)
V = acceleration voltage (constant)

Ions are sequentially brought into focus at the detector by scanning B

The machine is calibrated using samples with a known m/z

[Page 6]
Physical Biochemistry Mass Spectrometry 1

The magnetic sector is a scanning instrument e.g. 500-5000 m/z (would take 30s)
The consequence of this is that each specific value is only detectable for ~1/10s
For the other 29.9s the ion is non-detectable – its flight path will cause it to strike the magnet,
annihilating it.

§ Sensitivity: Low (as only a fraction of the ions are detected)

§ Upper mass range: Medium (~20kDa)
§ Resolution: Good (can be increased by adding an electrostatic analyser afterwards)
§ Ion transmission: Low
§ Expensive, requires a large magnet and a lot of space

II) Quadrupole
Uses a quadrupolar electric field (comprising of RF and DC components) to separate ions
§ Each diagonal pair of rods is connected electrically
§ One pair makes up the DC component
§ The other pair makes the RF potential
Consists of four parallel metal rods in a high vacuum chamber
The machine is calibrated using samples of known m/z to create a calibration curve.

1. The ions generated from the source try to pass through the quadrupole to the detector
2. Some ions (with a certain m/z) will be in resonance with the quadrupolar field
§ They will have a tight flight path, and will pass through the quadrupole, eventually hitting
the detector.
§ Non-resonant ions will be attracted to one of the poles, and will be annihilated upon impact
3. Scanning experiment through an m/z range

[Page 7]
Physical Biochemistry Mass Spectrometry 1

§ Upper mass range: Low (<4kDa)

§ Sensitivity: Low (as is a scanning type instrument)
§ Resolution: Low
§ Ion transmission: Low
§ Cheap, small and robust
§ Often used in GC-MS (EI ionisation) and ES-MS

III) Ion Trap

Use a similar principle to quadrupole, but do not operate as a filter
Ions are stored using RF fields (and sometimes DC voltages) applied to electrodes arranged in a
ʻsandwichʼ geometry.
§ Sandwich = a ring electrode with a cap on each end, leaving a chamber in the middle
Ions generated will pass into the central chamber, and are under the influence of a quadrupole-
like field.
The ions circulate within this chamber. The strength of the quadrupole field is then varied, which
causes ions of different m/z to exit the chamber (their circular paths become larger until they exit
from the hole. Initially ions with a low m/z will be ejected, then ions of higher m/z are sequentially
ejected.
Similar, but slightly better stats than quadrupoles

IV) Time-Of-Flight (TOF)

Ions are separated by differences in velocities as they move in a straight path towards the
detector. Ions with a smaller m/z will have a higher velocity, and so will hit detector faster than
ions with a higher m/z.
“Unlimited” mass range (as only measuring velocities – any mass as long as ion can fly)
Initially had poor resolution & mass accuracy

[Page 8]
Physical Biochemistry Mass Spectrometry 1

§ Ion transmission: Virtually all ions from source will pass down flight tube and be detected
§ Sensitivity: High, as almost all ions are detected
§ Cheap
§ Resolution: Used to be poor – delayed extraction in MALDI helped to improve this. Initial
kinetic energies and other factors need to be kept the same in all ions – reflectrons used.

Reflectron (ion mirror):

A series of electrodes that apply a potential at the end of the TOF flight tube
Ions are repelled by this potential, and will change direction
Ions of the same m/z, with different kinetic energy, will penetrate the reflectron to a different level
§ Ions with higher kinetic energy will penetrate deeper, causing them to slow down
§ They will hit the detector at the same time as other ions with the same m/z, but with a
lower kinetic energy
§ Some ions are lost (usually high MW ions)
Effectively, the reflectron corrects the kinetic energy issues.
Equipment without a reflectron is known as a linear analyser (resolution <1000), the reflectron
has a much higher resolution (>3000). A linear analyser might provide a broad peak, when a
reflectron is used this broad peak can be resolved into its internal peaks.
The length of the flight tube has been doubled, which also helps to increase the resolution.

V) Ion Cyclotron Resonance (FT-ICR)

Ions are passed into a metal box (trap)
The box is under a quadrupole field, as well as a massive magnetic field, close to absolute zero
§ Cooled using liquid helium
This causes the ions to circulate within the ʻcellʼ
A short burst of broad-band energy is added to the cell
§ Ions in the cell will pick up this energy and their flight paths will increase
§ This causes ions to come close to the top and bottom plates

[Page 9]
Physical Biochemistry Mass Spectrometry 1

§ The ions are charged, so they complete a circuit between the top and bottom plate,
leading to an electrical signal.
§ The RF or magnetic fields are scanned
§ A Fourier transformation of the time dependent signal is carried out, which can then be
converted into a mass spectrum

§ Fantastic performance
§ Virtually unlimited upper mass range
§ Ion transmission: Excellent – all ions are detected
§ Resolution: Highest
§ Very expensive, require huge superconducting magnets

VI) Orbitrap
Ions are trapped within a cell by static electrostatic fields (no magnetic field)
Contains an outer, and centre electrode (each with DC current – generating the electrostatic field)
Ions orbit around central electrode and oscillate in axial direction
m/z ratio relates to the frequency of ion oscillation along axial direction
The ions circulate and move up and down the electrode – the speed at which they do this is
related to the m/z.
Fourier transform algorithm converts the time-domain signal to m/z in order to compile the
spectra.

§ Very modern technique (2005)

§ Resolution: Very high
§ High mass accuracy
§ Ion transmission: Good
§ Upper mass range: Good
§ Cheaper than FT-ICR (no superconducting magnet)

[Page 10]
Physical Biochemistry Mass Spectrometry 1

Ion Detectors:
Orbitrap and FT-ICR detect ions inherently (no need for a detector)

PM (photon multiplier)
1. The ions enter the detector
2. The ions strike the converting dynode causing the
release of secondary electrons
3. The secondary electrons are attracted to a phosphorus
screen
4. The electrons strike the screen causing the release of
photons
5. Photons are finally detected by a photomultiplier tube

Several amplification steps occur

§ When the ion strikes the dynode it releases several secondary electrons
§ When the secondary electrons hit the screen several photons are released
§ The photomultiplier tube will also amplify the signal (105 x amplification)

EM (electron multiplier)
1. Ions are attracted into the cone, which is electrically grounded
2. The ion strikes the side of the cone
§ The inside of the cone has a special coating which leads to the release
of electrons
3. The electrons want to reach the grounding potential, so they cascade down
the tube
4. More electrons are then released (snowball effect)
106 x amplification

Most modern MS machines do not just have one ES, they have Multi Channel Plate (MCP) Array
detectors. These are several (100ʼs) of detectors arrayed on a plate. Increases the amplification
of the signal greatly.

Now that all of the parts of the MS machine have been listed, it is important to put them together
in the correct way; some parts work better with others for certain jobs.

[Page 11]
Physical Biochemistry Mass Spectrometry 1

EI:

The most common combination is an EI source with a quadrupole mass analyser

§ Very poor resolution and other stats
§ Cheap and small
EI and quadrupole match up well (similar properties)
§ EI can only ionise up to 1000 Da, so upper mass limit does not need to be higher
§ Resolution is dependent on m/z: if only using small molecules then high resolving power is
not required
§ GC (gas chromatogram) can be linked to EI as it uses gaseous samples. GC is a high
resolution separation technique

GC-MS is the method of choice for low MW samples

§ e.g. Odour and Volatile Organic Compounds (VOCs)
§ It is important to monitor air quality in many industries

1. Collect a VOC sample: air is pumped through a column containing an inert matrix, which will
capture the VOCs
2. VOCs are extracted from the matrix
3. Analysis using GC-MS

Very sensitive, can be used in forensics. Smoke / air has an MS ʻfingerprintʼ

FAB:

Fast atom bombardment ionisers are most commonly found on magnetic sector instruments
§ Matched upper mass ranges
§ Magnetic sector is a slow, scanning instrument that can miss out ions due to the limited
time for each m/z
§ FAB ionisation can be used for ~5 mins allowing longer scans, increasing the sensitivity
for detection
§ Expensive and large, very few still used or made

MALDI:

MALDI sources are most commonly found on TOF instruments

§ MALDI can use large molecules, TOF is unlimited in terms of mass
§ The laser pulses produce tight ʻbunchesʼ of ions

[Page 12]
Physical Biochemistry Mass Spectrometry 1

§ TOF analyses need tight bunches of ions

§ TOF detects all ions produced, increasing sensitivity (cf. FAB magnetic sector)
§ Found very commonly in analytical labs e.g. petrochem industry, forensics

ES:

ES sources are commonly used with most mass analysers

Can be used with quadrupole despite the fact that quadrupole cannot handle ions with high mass
Quadrupole separates on m/z (ES produces multiply charged ions)
The m/z can be reduced significantly (20+ charge states can be reached)

FT-ICR can be fitted with all types of sources, but they are expensive so not common.

Hybrid Instruments:

Many modern MS machines utilise a combination of mass analysers within the same instrument
(rather than a single mass analyser)
§ MALDI–TOF–TOF
§ MALDI–Quad–Ion Trap–TOF (good resolution)
§ ES–LTQ–Orbitrap (High resolution)
§ ES–Quad–FT-ICR (Extremely high resolution)
All have femtomole sensitivity

[Page 13]
Physical Biochemistry Mass Spectrometry 1

Analysis:
EI ionisation is from gaseous phase (most biomolecules are not)
Mass spec is very useful for solving the structure of biomolecules
Most biomolecules are polymers (DNA, RNA, Proteins, Complex Carbohydrates)
The complex molecule has energy added, and it is ionised
This will produce molecular ions (all components of biomolecule)

Molecular Ions
Different ionisation techniques will produce different types of molecular ions
§ EI: Radical cation; [M+.] (true molecular ion)
o Mass of an electron is minimal, so in an EI experiment the mass of the molecular ion
should be equal to that of the molecule
o No complete molecular ion peaks are seen, as EI causes fragmentation (apart from
certain molecules with many rings / double bonds which can help to delocalise energy)
o e.g. Alkaloids
§ FAB, MALDI: Singly charged cations and anions; [M+H]+, [M+Na]+, [M-H]-
o A counter ion is picked up (pseudo molecular ions)
o m/z does not equal mass of molecule (need to take counter ion into account)
§ ES: Multiply charged cations and anions; [M+nH]n+, [M+nNa]n+, [M-nH]n-
o Negative charges can be caused by losing multiple H or picking up multiple Cl

Nominal Mass & Accurate Mass:

Masses are relative to 12C, which has a mass of exactly 12.

1
H has a nominal mass of 1, however it has an accurate mass of 1.00783
The same goes for 16O, 14N, and 13C
Some atoms will be slightly heavier / lighter than expected

Accurate mass = 400.0000

Measured mass = 400.0020
Difference = 0.0020 or 2 mmu (milli mass units)
Relative mass accuracy: Error = 0.002/400 × 106 = 5 ppm (part per million)
Greater error = greater number of possible formulae
Larger samples amplify the effect - ↑ more possible formulae
Orbitrap < 2 ppm (high accuracy)

[Page 14]
Physical Biochemistry Mass Spectrometry 1

Resolving power is the ability of an MS

machine to separate two peaks up to
10% of the peak height. High resolving
power is required to distinguish between
nominal / accurate mass.

RP required to separate signals =m/Δm

N2 (28.0062)
CO (27.9949)
Difference = 0.0113

RP = 28/0.0113

A resolving power of 2,400 is required

If the nominal mass was 280, with the same difference, a resolving power of 24,000 would be
required to separate the peaks. Quadrupole has a resolving power of 4000 – could not resolve
these peaks, use orbitrap / FT-ICR instead.

Isotopes
13
C accounts for around ~1.1% C atoms
Organic molecules often contain a lot of carbon
If a peptide has 100 carbons, you can expect it to contain at least one 13C
In reality, ʻidenticalʼ samples contain a combination of different isotopes
Often there will not be a single molecular ion peak, there will be a cluster of peaks (isotope
clusters) due to the 13C. Peaks >2000 are very likely to contain at least one 13C
Average mass – when RP is not high enough to see different isotopes (relates to whole cluster)

e.g.
@ 2000 (m/z): most abundant peak has one 13C
@ 5000 (m/z): most abundant peak has two 13C
Each peak is 1 mass unit apart

Isotope clusters are useful for providing atomic

information. Higher RP required at greater masses

2
H (D), 15N, 17O, 33S and 34S are all naturally occurring heavy isotopes

[Page 15]
Physical Biochemistry Mass Spectrometry 1

Derivitisation:

The best way to do this is by derivatising these molecules – decrease interaction that make the
molecule a liquid / solid (e.g. H-bonding, salt bridges etc.) And make more hydrophobic.
1. Acetylation of amino and hydroxyl groups using acetic anhydride: (CH3CO)2O
§ Reduced H-bonding

2. Trimethylsiylation – adding a TMS group to a esters, ketones etc.

o More hydrophobic

3. Ketones → ketals

4. Methylation (-CH3) of hydroxyl, amide, thiol etc. Less H-bonding

5. Esterification of acids

[Page 16]
Physical Biochemistry Mass Spectrometry 1

Hydrophobic molecules tend to ionise better than hydrophilic molecules, increased sensitivity of
detection.

EI Fragmentation Pathways:
EI has enough energy associated with it to fragment a molecular ion
All fragmentation pathways are unimolecular (no additional chemical groups are introduced)
§ Molecular ion is fragmented, and fragments can be further fragmented
MS can only detect charged species, neutral species will be lost

Radical Elimination

Radical ions can eliminate radicals of neutral molecules

A+. → B+ + C.
A+. → B+. + C
C will not be seen in either case, as it is neutral. Only B will be seen in the mass spectrum.
Reaction initiation at the radical site arises from its strong tendency for electron pairing, the odd
electron is donated to form a new bond to an adjacent atom. This is accompanied by cleavage of
another bond to that α-atom

Even electron rule: an even electron ion will not normally fragment into two odd electron species

[Page 17]
Physical Biochemistry Mass Spectrometry 1

Nitrogen, as well as O / S are good atoms for potentially localising radical / charge.
The electron in a radical wants to form a new bond (single headed arrow denotes single e-)

Neutral Elimination
Occurs when a highly stable product can be formed from the fragmentation reaction
This is a common fragmentation in all types of MS
Remember that all MS reactions are unimolecular - do not use acid/base
e.g. alcohol → ethylene

H2O not seen as it is not charged

Inductive Cleavage
The driving force is the molecule trying to neutralise a positive charge
Both electrons from a bond are moved towards the positive charge
A neutral radical and a new cation species are generated
The charge migrates from the oxygen to the alkyl group (in the example below)

[Page 18]
Physical Biochemistry Mass Spectrometry 1

McLafferty Rearrangement

As this is high-energy gas phase chemistry, entire groups can me moved around
Bonds break then reform, but only the atoms from the original molecular ion can be used
Rearrangement involves transfer of an H from an atom which is γ to an O/N/S
Occurs with esters, carboxylic acids, amides etc.

Example – Ketone-like functional group

A pseudo six-membered ring is formed between γ carbon and the O atom

Electrons can be moved around this pseudo ring
A proton on the γ carbon migrates onto the oxygen
A new product with a hydroxyl group is formed

Fragment ions are created by very well defined fragmentation pathways

Only EI and FAB produce fragment ions from the initial ionisation step (MALDI, ES etc. do not)

MS/MS (Collisional activated decomposition) Experiments:

Requires a setup with at least two mass analysers in tandem
Between the two mass analysers there is a collision cell
Within the collision cell there is a small amount of an inert gas (He, Ne etc.)
1. Sample is ionised
2. Ions are separated in the 1st mass analyser
3. A component of the 1st mass spectrum is selected
4. This component is passed onto the collision chamber
5. Ions collide with inert gas molecules
6. Collision provides a second input of energy that is sufficient to cause the induction of
fragmentation within the molecular ions (some molecular ions will not be fragmented)
7. Fragment ions leave collision cell and enter the 2nd mass analyser, where they are separated
by m/z and subsequently detected

[Page 19]
Physical Biochemistry Mass Spectrometry 1

Triple Quadrupole
The first MS/MS technique used

ES → Quad1 → Quad2 → Quad3 → Detector

Quad1 & 3 – Mass analysers

Quad2 – Collision chamber

This allows fragment ions to be generated from an ES or MALDI type experiment

Low resolution, sensitivity and upper mass range. These drawbacks are multiplied due to there
being 3 quadrupoles.

Q-TOF (Quadrupole Orthogonal Acceleration Time-Of-Flight)

3rd quadrupole was replaced with a TOF analyser & reflectron

ES Source
1st Quadrupole
2nd Quadrupole (collision cell)
Reflectrom TOF

Allowed better resolution and

sensitivity as all ions are detected
(not a scanning instrument)
Very useful for proteomics.

ES-MS/MS Peptide Sequencing:

Advantages of using ES
§ It can be set up with liquid chromatography (for separation)
§ Very good at ionising peptides
§ Generates multiply charged ions, which can have advantages in proteomics. They tend to
be easier to fragment than singly charged ions

[Page 20]
Physical Biochemistry Mass Spectrometry 1

§ Peptides will tend to have multiple charges whereas the general chemical background will
usually be singly charged. This allows isolation of the peptides reducing the signal : noise.
§ Multiply charged ions are readily recognised by the interval between isotope peaks

A good way of getting multiple charges onto peptides is to generate the peptides using trypsin
§ Cleaves after C-terminal arginine (R) or lysine (K)
§ Tryptic peptides have a minimum of 2 charges:N-terminus +, C terminal + (K/R)

Recognising multiply charged ions

Involves 13C
Peptides do not give single peak, they give an isotope cluster

M = 1000 Da

[M+H]+: m/z = 1001 / 13C m/z = 1002 (separation = 1)

[M+2H]2+: m/z = 501 / 13C m/z = 501.5 (separation = 0.5)
[M+3H]3+: m/z = 334.33 / 13C m/z = 334.66 (separation = 0.33)

By looking at the difference between nearby peaks (<1 m/z away) we can determine whether the
peak refers to a singly or multiply charged fragment.
Once an isotope cluster has been identified, it can be passed into the collision cell, where
fragmentation will be induced. A fragment ion spectrum can be generated from the cluster.

Fragmentation of Peptides
Occurs by highly defined fragmentation pathways (collisional induced dissociation)
This allows easy analysis of the MS/MS spectrum, and for the peptide sequence to be derived
Ends are important as peptides are asymmetric
A peptides mass will be the sum of its residues, + 1 mass unit at the N-terminus (H–) + 17 mass
units at the C-terminus (–OH).
The counter-ion must also be taken into account e.g. H (+1)
In this case it would be the sum of the residue masses +19.

b-ion fragmentation pathway

Fragmentation from the N-terminus towards the C-terminus of a peptide
b-ions = residue mass(es) + 1 (proton)

[Page 21]
Physical Biochemistry Mass Spectrometry 1

N atom has quaternary valency and a positive charge. This is the driving force for the
fragmentation reaction.

Additional fragmentation can occur to produce a-ions

Neutral elimination occurs (eliminating CO)

a-ions have a mass of 28 less than their parent b-ion, due to the loss of CO

[Page 22]
Physical Biochemistry Mass Spectrometry 1

y-ion fragmentation pathway

Starts from the C-terminus, moving towards the n-terminus, generating c-terminal y-ions

y-ion mass = residue mass + 17 (-OH) + 2 (2 × H)

The spectrum will contain a series of b-ions, a series of y-ions and perhaps some a-ions, from
which the amino acid sequence can be determined.

MS has been at the centre of modern ʻomicsʼ techniques (proteomic, glycomics, metabolomics)

[Page 23]
Physical Biochemistry Mass Spectrometry 1

Proteomics:
Proteins are the most important biomolecules as the ʻdo thingsʼ
§ What proteins are expressed and when?
§ What amounts?
§ What modifications occur? (phosphorylation, glycosylation etc.)
§ How do they interact?
§ Do levels change during differentiation / disease?

2DGE (2-D Gel Electrophoresis)

The most widely used separation technology fir proteomics
Separation by electric charge on one axis, MW on the other

Advantages
§ Quick (~2 hours)
§ Cheap
§ Good at resolving complex mixtures
§ Crude samples are OK (can use cell homogenate)
§ Moderate salt / detergent tolerance
§ Good visual representation of entire sample (stain & make digital images)
§ Easy comparison
§ Relatively reproducible (use precast gels, which are very similar to one another)

Stains (coomassie blue, silver stain), radiolabelling and fluorescent molecules can be used for
detection on the gel. Will show proteins as spots on the gel. MS can then be used for rigorous
identification.

The proteins that are changing in

abundance are the proteins that are
most likely to be involved in the disease.

These proteins could be targets for

drugs.

The proteins are identified using MS

[Page 24]
Physical Biochemistry Mass Spectrometry 1

Proteomics workflow
1. Run 2D gel
2. Identify spots that have been up/down-regulated
3. Excise spots
4. Carry out in-gel tryptic digest
5. Tryptic peptides are eluted
6. MALDI-TOF MS carried out on tryptpic peptides
7. Peptide sequence can be determined
8. Sequences are searched for in a database, look for matched
§ Computers carry out in silico tryptic digests for the entire (coding) human genome to
generate potential peptide database
§ Hopefully find good sequence identity which will lead you to a protein

If genome of organism has not been sequenced, or protein has had post-transcriptional
modifications (might cause computer program to miss the peptide) then a higher level of
structural information is required.

MS/MS experiments allow greater insight into structural information

e.g. NanoLC-ES-MS/MS
On-line Nano liquid chromatography (Reverse phase, ion-exchange etc.) prior to MS allows more
separation before the MS step and can give insight into post-translational modification

Even in unsequenced genomes, there is still often a great deal of similarity between related
proteins in other species.

Glycomics
Determining the glycan repertoire in a cell / tissue / organ as a 1st step to defining function
Prior knowledge of glycan biosynthetic pathways is essential
Glycosylation is the most common post-transcriptional modification

Extraction chemistry can separate glycolipids and glycoproteins

§ N-linked glycans can be released using the enzyme PNGaseF
§ O-linked glycans can be released using a chemical elimination method
§ Glycolipid derived glycans can be released enzymatically using endoceramidase

Original starting material → N-glycans + O-glycans + glycolipid derived glycans

Derivisation increases sensitivity of detection of glycan
Permethylation reaction is carried out on all released glycans

[Page 25]
Physical Biochemistry Mass Spectrometry 1

MS can then be carried out on the glycans: MALDI-TOF, ES-Q-TOF, GCMS-EI

Hexose sugar: Glucose, Mannose, Galactose

Deoxy sugar: Fucose
N-acetyl: N-acetylglucosamine, N-acetylgalactosamine,
Sialic acid: N-acetylneuraminic acid

The hexose sugars are structural isomers, so MS cannot differentiate between them
The same goes for the N-acetyl molecules
Peptides are linear, but glycocidic bonds are not (α/β bonds, multiple linkage positions)
Knowledge of N/O glycan biosynthetic pathways helps us to overcome this

N-glycosylation starts with a single precursor: 3 Glu, 9 Man, 2GlcNAc

Modification then occurs (as above) in a highly ordered way, due to the high specificity of
different glycosyltransferases and glycosidases
In a human system we know all of the enzymes in this pathway

[Page 26]
Physical Biochemistry Mass Spectrometry 1

O-glycosylation is initiated by the addition of N-acetylgalactosamine

Can be extended by a Gal to make a core 1 structure, which can then be sialated, or antennas
can be extended or add a N-acetylglucosamine
The enzymatic pathways for O-glycosylation are known

A methylation reaction converts the highly hydrophilic glycans into more hydrophobic molecules
Sugars have many –OH groups which can be methylated to –O–CH3
Fucose has 2 –OH groups that can be methylated
Hexose residues have 3 –OH groups
NAc residues can have their –OH groups methylated, as well as the N in the N-acetyl group
HexNAc – 3 methylation events
Sialic acids – 5 methylation events

Carbohydrates are asymmetric; they have a non-reducing end and a reducing end
In MS experiments on carbohydrates, ends have to be taken into account
Non-reducing end: Methylation will add 15 for a CH3 group
Reducing end: 16 + 15
Total ends: 46 mass units
The ion being used to generate charge needs to be taken into account too – usually for MALDI
spectra a [M+Na]+ (singly charged ion) so add 23 on top.

The quality & sensitivity of the spectra after permethylation is greatly increased (↓ signal:noise)

Glycomics can be used to look at changes from a healthy situation to a diseased situation, like in
proteomics. Efforts are being made to characterise biomarkers for a range of diseases, especially
cancers.

Although molecular ions give us a decent amount of information, generating fragment ions
massively increases molecular structure.

MS/MS can be carried out on glycans (ES-Q-TOF, ES-ion trap, MALDI-Q-TOF etc.)
MALDI-TOF-TOF is potentially the most powerful for automated high-throughput sequencing
Glycans have highly defined fragmentation pathways

[Page 27]
Physical Biochemistry Mass Spectrometry 1

Metabolomics
Determining the small molecule metabolite repertoire in a biological system (cell, tissue, body
fluid etc.) as a result of its metabolism.
Important to find out how drugs are metabolised in the body e.g. production of toxins

1. Extraction
2. Purification
3. Derivitisation (to increase sensitivity) e.g. TMS reagent
4. MS analysis (GC-MS, LC-MS, LC-MS/MS, EI-GC-MS – Small MW molecules so high mass /
resolution not required. MW ~1000 Da, so OK to use cheaper techniques)
§ GC step prior to MS is advantageous
5. Data analysis

High sensitivity MS is able to deal with very complex mixtures such as urine, which contains
1000s metabolites

[Page 28]