For a second year Imperial College student studying Biochemistry
Content is detailed & extensive.
Content includes:
- Mass spectrometry (pros/cons, parts, mass spec data, ionisation sources, mass analysers and their performance characteristics, TOF analysers, hybrid instrument)
- Peptide seq...
Mass Spectrometry
A mass spectrometer is an instrument which is used to define the covalent structures of substances by
ionizing, separating, and detecting molecular and fragment ions according to their mass-to-charge ratios
(m/z).
Advantages:
MassSpec is extremely sensitive and so can be carried out on very tiny amounts of material eg
femtomoles or less (10-15 mole) and can be used to study very complex mixtures eg urine extracts,
perfumes, protein digests etc. it is a LOT more sensitive than NMR.
It can also be used to study mixtures of sample. So you don’t have to have highly purified, homogenous
samples to get good quality data, unlike Xray Crystallography. You do not need to purify proteins.
The three basic parts of a mass spectrometer are:
Ion source: Creates charged ions (free radicals)
Mass Analyzer: Separates them based on their mass to charge ratio
Detector: Detect the signals associated with them to get quantitative information about which
components of our mixture are present in a high and low abundance.
What does Mass Spectrometry data look like?
Both the intensity and mass/charge ratio do not have specific units attached to them.
The tallest peak (highest intensity) represents the most abundant component in the mixture, it has the
highest ion count. This component is then given an arbitrary component of 100% relative intensity and
therefore all the other components in the spectrum are expressed as a relative percentage of the most
abundant peak in the spectrum.
Ionization (Ion) Sources
What happens:
1. High energy electrons come into close enough proximity with the gas phase molecules so that the
electron in the outer orbital of our biological molecules is repelled by the negatively charged
electron beam out of the orbital.
2. This loss of electrons from the outer orbital results in a positively charged cation with an unpaired
electron in its outer orbital. Hence, this cation is now a free radical.
Different Ionization Methods in the Ion Source
Here are the 3 most common methods:
• Electron Impact (EI)
o Described as a “hard” ionization technique which means it has high energy. Because of this,
there can be an excess of energy given to the biological molecules.
o Once the sample is ionized, additional energy can then lead to fragmentation of the gas phase
ions which is useful for structural elucidation.
▪ Gas phase sample is bombarded with high energy electrons coming from rhenium or
tungsten filament when they are heated (energy = 70 eV).
▪ Larger potential difference = Higher energy during collision
, ▪ Only produces positively-charged ions [1]
▪ Produces mostly fragment ions [1]
o The average bond in a biological molecule is about 5eV so there is a large amount of excess
energy associated with the electron beam. This excess energy fragments bonds. Most of the
molecular ions decompose into fragments (70 eV >> 5 eV bonds) via uni-molecular reactions.
o Commonly combined with Quadrupole mass analyzer [1]
o OUTSIDE READING:
Why 70eV?
The ionization cross section plot shows the probability of ionization as a function of
electron energy (eV). The higher the signal, the better the odds. that 70eV is
essentially the “best” value.
This graph shows that the
theoretical and experimental
model maps very closely at
70eV. This is the maximum
ionization efficiency.
70eV gives consistent
ionization for quantitative
analysis. We want the signals
to change due to concentration
and not due to the mass spec.
70eV
o Good at ionizing small molecules, 1-1000 Da
▪ Bc smaller molecules convert to gas phase easier and EI ionization occurs in the gas
phase
▪ Generally, you can create gas phase ions by heating the sample up from a probe tip until
it evaporates or from an online gas chromatograph.
▪ Because your analytical molecules are in the gas phase, it is quite common to link the EI
to a Gas Chromatography Separation System. So you get your molecules into the gas
phase and separate the components of the mixture on an online gas chromatography
initial experiment and then do the electron impact ionization and mass spectrometry
characterization.
o Lowest ionization cutoff
o What happens:
1. First, your gas phase sample enters into the EI source.
2. There is a heated filament that produces the electron beam and magnets on either side to
focus the beam into a tight band, thus increasing the chances of ionization of the biological
sample.
3. Once the gas phase actually become gas phase IONS (M+.) aka radicals, you can control the
movements of these ions. At the back of the EI source, you have an “Ion repeller” with a
, large positive potential that will repel the positively charged cation M+. and lead them to
move towards the MassSpec for future analysis.
• Electrospray Ionization (ES or ESI – Soft)
o “Soft” ionization technique because JUST enough energy is provided to the biological molecule
to convert it to gas phase ions such that there is no excess energy leading to fragmentation.
o For larger biological molecules such as peptides, oligosaccharides, proteins. For molecules
greater than 500,000 Da
o This technique is conducted at atmospheric pressure
o Ionizes from Liquid Phase
▪ This is an advantage bc most biological molecules occur in the liquid phase.
▪ Starting from the liquid phase allows us to link up different types of liquid
chromatography systems to our mass spectrometer. So we can start with a complex
mixture of biological molecules, separate them on an online liquid chromatography
column (like Reverse Phase) and then inject and spray individual separated components
from the chromatography into the mass spectrometer.
o Can generate positively- and negatively charged ions but only analyze one type at a time. We
can control either the positively- or negatively charged ions by controlling the electrical
potential on the plates surrounding the ions when they’re generated.
o Advantage: search “Advantages of ES (Electrospray) Ionization”
o What happens:
End of needle
covered in metal
coating
1. We take our biological sample of interest, dissolve it in a suitable solvent then apply that
solvent to a small capillary needle.
, 2. The end of the glass capillary needle has a tiny hole and the outer layer of the needle is
covered in a metal coating. The narrow glass capillary’s tip is coated with gold.
3. This needle carrying our sample is introduced into a “covering electrode”.
4. Because we’ve got a metal surface on the end of our needle, you generate a very large
electrical potential between the end of the needle and the electrode.
5. When we put a little pressure on the tip of the needle (A high voltage (3-4 kV)), you force
your sample out of the needle and the sample emerges from the tip and is dispersed as an
aerosol of highly charged droplets.
6. As the droplets move away from the electrode and towards the mass spectrometer, they
come across a dry gas.
7. Drying gas (nitrogen) flows around the outside of the capillary to evaporate the solvent
components of your droplets, decreasing the size of the droplets.
8. As the solvent evaporates, the droplets get smaller, electric field increases and droplets
become unstable due to high surface charge and they repel and move to the surface.
9. Finally, a point is reached where there is not enough solvent to keep similarly-charged
peptides ions separate and the droplets will “explode” into smaller droplets. This is the
Rayleigh Limit which is the Point at which the volume of the drop is too small to contain all
the charged ions within the drop that want to repel each other
10. The process continues until you reach a point where all of the liquid solvent is evaporated
and you’re just left with “naked” gas phase peptide ions whose charge corresponds to the
charge of the original molecule at the pH of the experiment (other factors).
11. These ions drift into the analyser because of the voltage gradient.
o Advancement: Nanospray
▪ Early ESI sources operated at flow rates of a few µl/min. As technology developed, the
flow rates were dramatically reduced to nl/min (10-30 nl/min). These are called
“Nanospray”.
▪ NanoES is carried out with “needles” into which about 1 ml of a solution of the sample is
added - therefore time(?) for many MS and MS/MS experiments on a single sample.
▪ Advantages:
- Requires a lot less starting material to generate your spectrum, thus increasing
the sensitivity of the electrospray experiment.
• Matrix Assisted Laser Desorption Ionisation (MALDI)
o Soft ionization technique (meaning the molecular ions remain intact when ionized)
o Also for bigger biological molecules such as peptides, proteins, DNA, up to 500,000 Da
o Occurs from the solid phase instead of the gas phase
o MALDI Lasers
▪ Nitrogen gas UV laser
- Wavelength 337nm
- Repetition Rate 1-20Hz
- Pulse energy 120-1200µJ
▪ Solid State UV Laser
- High repetition rate
- Wavelength 355nm
- Favored and newer
- A Nd-YAG laser
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