Mass Spectrometry Flashcards
Uses of Mass Spec.
Drug Testing
Quality Control
Identification of Proteins
Forensics
What does Mass Spec. measure?
Measures the mass/charge ratio (m/z)
Electron Ionisation Mass Spectrometer (EI-MS)
A hard ionisation technique (i.e. causes fragmentation of molecules)
Useful for small molecules but not for macromolecules.
Sample is vaporised and bombarded with electrons, causes a charged species with +1 charge
A magnetic field separates particles based on their mass/charge ratio.
Distances between isotopic peaks on spectrum reveal charge state.
2 Soft Ionisation Techniques
Electrospray Ionisation (ESI)
Matrix-Assisted Laser Desorption Ionisation (MALDI)
Cause significantly less fragmentation of molecules because they impart little residual energy onto the subject molecule. This makes them well-suited for studying larger molecules.
Electrospray Ionisation (ESI)
A soft ionisation technique:
1. Ionises a continuous flow of molecules in solution
2. Produces multiply charged ions (e.g. [M+nH]^n+)
3. Observed charge envelope characteristic of the number of ionisable sites (i.e. basic sites will be protonated and acidic site deprotonated)
ESI is easily coupled with separation techniques such as High-Performance Liquid chromatography
LIKELY EXAM QUESTION ON THIS:
n=[(mn+1) - H]/(mn)-(mn+1), and then M = [(mn) X n] - n
Note:
Always round n to an integer since it represents a charge
n on right of spectra smaller than n on left of spectra
M is the mass of an uncharged molecule
Use above formulas to calculate. Best to calculate for all given peaks and then average, to get the most accurate result.
Matrix-Assisted Laser Desorption Ionisation (MALDI)
A soft ionisation technique:
- Analyte is co-deposited with Matrix (a small molecule which absorbs photon energy and transfers it to the analyte)
- Laser excites matrix which transfers energy to analyte.
- Produces predominately singly charged species i.e. [M+H]+
- Typically used for large biomolecules
- MALDI is a high mass/pulsed source so usually combined with TOF (a mass analyser)
- Very sensitive (at attomole (10^-18) level) but less sensitive to contaminants such as salts and detergents
- High throughput analysis (up to ~3000 samples/day)
- Samples can be re-analysed.
-Can ionise high molecular weight species
DISADVANTAGES:
-Coupling MALDI with chromatography is difficult.
-Pulsed nature of source limits compatibility with many mass analyzers.
-MALDI matrix cluster ions obscure low m/z (<600) range.
-Analytes that absorb laser light can be problematic.
Mass Analysers
Time-of-Flight (TOF) Quadrupole (Q) Ion trap (IT) Orbitrap Ion Cyclotron Resonance (ICR)
Factors affecting choice of mass analyser
Mass range Resolving power Mass accuracy and ease of calibration Speed Scanning or non-scanning? Compatibility with with ion sources Ease of use for tandem mass spectrometry
Time-of-Flight (TOF)
- Fast, sensitive, simple, cheap
- Electrostatic “mirrors” (Reflectrons) used to lengthen flight time and reduce spread of ion populations → higher resolution
Quadrupole (Q)
-Four parallel metal rods, ions oscillate and at a specific U/Rf only a certain m/z passes through the quadrupole. By continuously varying U/Rf, the Q can be used for scanning.
U = DC potential, Rf = Radio Frequency
Small and portable, but low resolution (~2000)
Ion trap (IT)
- Similar to quadrupole – but in 3D
- 2 common forms: Linear Ion Trap (LIT) and 3D/Paul Ion Trap (QIT)
- Resolution 2,000-10,000 depending on scanning speed
Orbitrap
- Ions are injected into the electric field between electrodes and trapped. Ions oscillate around and along the inner electrode according to the m/z.
- Acts as mass analyzer and detector
- Sensitive, high resolution (<500k), high accuracy
- Requires “C-trap” to inject tight ion packages.
Fragmentation Techniques
Peptides can be fragmented using different techniques yielding distinct but predictable cleavage fragments:
- Collision-Induced Dissociation (CID)
- Electron-Transfer Dissociation (ETD)
- Higher-energy Collision (C-trap) Dissociation (HCD)
a and x cleave between R group and carbonyl
b and y cleave between carbonyl and NH
c and z cleave between NH and R group
Top-Down proteomics
Individual species can be more exhaustively characterised in their entirety. Doesn’t fragment the protein, keeps protein intact. However is restricted to simple protein mixtures.
Bottom-Up proteomics
Enables the identification of all proteins in highly complex samples. Cuts proteins into fragments with a protease.
