Protein Mass Spectrometry Flashcards
What is mass spectrometry?
It measures the m/z ratio of a substance by mass and charge separation.
True or false:
Mass spectrum isn’t very accurate at determining mass
False
<0.01% error
True or false:
Mass Spectrometry can be used to determine structure
True
Only certain ions will hit the detector, depending on the _ force
Centripedal
Mass spectrometry occurs in a _
Vacuum
What is the M+ peak
A peak of 1 higher than the mass
Caused by C-13
Protein ionisation methods
Electron impact
Fast atom bombardment
MALDI and ESI
Electron impact
Causes extensive fragmentation so can only be used on substances <4000Da
Not suitable for proteins
Fast atom bombardment
Sample is in a liquid matrix and is bombarded by noble gases.
Soft ionisation method, so causes little fragmentation.
However causes selective desorption so is less commonly used.
Matrix Assisted Laser Desorption Ionisation (MALDI)
Analyte is mixed with excess organic matrix dried onto a support.
The matrix and the analyte are vapourised.
Matrix absorbs UV laser energy, but not analyte.
H+ transfer ionises analyte
Pros of MALDI
Leaves large macromolecules intact and single charged.
Electrospray Ionisation
50:50 H2O to Organic
Produces multiply charged ions so heavier molecules are possible.
Ions are sprayed out and enter drying gas
Coulombic explosion results in all single ions
Should you use MALDI or ESI for core complex samples?
ESI
Separation methods
Quadrupole
Ion Trap
Time of Flight
Quadrupole
There is alternating potential in 4 rods
Ions of particular m/z in resonance
Ions of unstable trajectory don’t reach the detector.
Quadrupole Ion Trap
3 sets of electrode surrounding a cavity
Ion orbit depends on V and m/z
Ions are released in increasing m/z by gradual change in potential
Can trap single m/z
Time of Flight
Ions are given a pulse of energy
Smaller ions = higher velocity = arrive first
Unlimited mass range, but worse resolution
Because we can’t tune for ions they must all be released at the same time.
Examples of paired ionisation and detection methods
MALDI-TOF
ESI-Ion trap
What is tandem mass spectrometry
Two mass spectrometers one after the other
Uses on spectrometer to analyse the fragments produced by the other
Produces a simpler easier to interpret spectrum.
Sequencing by fragmenting at fixed points
Breaking peptide bonds at the weakest point, N-O
One fragment has C terminus, the other the N
o A, b, c if contain N terminus
o X, y, z if contain C terminus
B and y are breaks on the peptide bond – most common
Sequencing by Incremental m/z
We know the mass of each amino acid, so we can use them to tell the differences between peaks.
e.g: Gly = 57 Da, Thr = 101 Da, Val = 99.1 Da -
Some are same so must break down side chain.
Uses of Protein Mass Spec
- Often for sequencing
- Detecting modified proteins – very good at this
Glycosylation detection
Can detect certain peaks that are a specific amount to big
e.g: sialic acif is 293
How can Mass Spec be used to determine 3D structural information?
By digesting proteins such that a specific linker remains.
Can then sequence the products ti find out which lysines for example are close together in space.
