mass spectrometry and proteomics - lecture 2 · tandem mass spectrometry • ms/ms, ms2 or tandem...
TRANSCRIPT
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Previously:
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Electrospray
MALDI
Resolution and other basics
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Lecture 2
• Mass analysers• Detectors• Tandem mass spectrometry
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Mass analysers
• Sector instruments (magnetic- B; electrostatic – E)• Time-of-Flight (TOF)• Quadrupol (Q)• Ion Trap (IT)• Ion Cyclotron Resonance (ICR)• Orbitrap
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Factors affecting choice of Mass analyser
• Mass range• Resolving power• Mass accuracy and ease of calibration• Speed• Scanning or not scanning?• Ease of interfacing with ion sources• Ease of use for tandem mass spectrometry
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Sector instruments
• Mass range up to m/z 15,000• Resolving power up to 100,000• Most suitable ionisation: EI, CI,
FAB, FD• Less suitable for ESI or MALDI• Rarely used nowadays, large,
complicated
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Time-of-Flight (TOF)
• Requires pulsed ion packages (MALDI, pulser in ESI-QTOF)• Ions travel in a field-free region according to their kinetic energy
Ek=mv2/2; i.e. mass can be determined if speed is measured.• Theoretically unlimited mass range (realistically >250k)• Resolving power up to ~30,000• Electrostatic mirrors (Reflectrons) used to lengthen flight time and
reduce spread of ion populations higher resolution• Fast, sensitive, simple, cheap
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Better resolving power: Reflectron TOF
D e te c to r
Ion source
Acceleration region Reflectron
Field-free drift region
Ion sourceReflectron
Detector Reflectron
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Time-of-Flight (TOF)
©Agilent
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Quadrupole (Q)
• Four parallel metal rods, opposite rods have DC potentials (U) on which a radio frequency is superimposed.
• Ions oscillate and at a fixed U/Rf only a certain m/z passes through the quadrupole. By continuously varying U/Rf, the Q can be used for scanning.
Ion motion in a Quadrupole
Quadrupole schematic
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Quadrupole (Q)
• Advantages: small, light, portable; robust; no high voltage; high dynamic range; cheap
• Disadvantages: low resolution (~2000); mass range m/z <3,000
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Miniature Mass Spectrometers
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Ion Trap (IT)• Two commonly used forms exist: Linear Ion Trap (LIT – e.g. Velos,
Thermo) and 3D or Paul Ion Trap (QIT – e.g. amaZon, Bruker)• Similar to quadrupole – but in 3D.• Equations of ion motion are complex.• Space charge effects possible when many ions in trap.• Resolution 2,000-10,000 depending on scanning speed.• Mass range <2,000, can only scan down to 1/3 m/z of precursor in MS/MS loss of low molecular masses.
Linear IT
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3D Ion Trap (QIT)
3D IT
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Potential well and ion motion in 3D trap
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Fourier Transform Ion Cyclotron Resonance (FT ICR)
• The m/z is determined based on the cyclotron frequency of ions in a fixed magnetic field (requiring superconducting magnet).
• Ions are trapped in a Penning Trap (ion trap in magnetic field) and excited to a cyclotron motion by an oscillating electric field.
• Very high mass accuracy (<0.5 ppm) and very high resolution (>1M)• Ions are not destroyed thus time dependent studies such as gas
reactions in the ICR cell are possible.• Very expensive in purchase and maintenance, tricky to run
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FT-ICR MS – working principle
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Cyclotron frequency of ion:
http://www.chm.bris.ac.uk/ms/theory/fticr-massspec.html
m2zBωC
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Orbitrap
• Developed in 2000 by Alexander Makarov, commercialised in 2005 by Thermo.
• Ions are injected into the electric field between electrodes and trapped. Ions oscillate around and along the inner electrode according to the m/z. FT allowsmeasurement of mass.
• Requires ultra-high vacuum (10-10 bar)• Requires “C-trap” to inject tight ion packages.• High mass accuracy (<1 ppm), high resolution (<500k)• Sensitive and high dynamic range.• Much cheaper than FT ICR.
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The Story of the Orbitrap
57The Analytical Scientist , Nov 2013
Alexander Makarov, 1990s
Alexander Makarov with Orbitrap2013
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Ion Mobility Spectrometer
• Ions are separated by shape, not m/z.• Drift gas and electric field opposed or orthogonal.• Drift time increases with collisional cross-section.• Often combined with mass spectrometry.
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Ion mobility spectrum
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Kloniecki et al, 2011
Oligomers of Alzheimer’s disease Aβ1-40 form several different structural states.
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Detectors• Commonly used are Secondary Electron multipliers (SEM),
Channel electron multipliers (CEM) and Microchannel Plates (MCP).
• These work by the principle that energetic particles hitting a semiconductor or metal surface lead to emission of secondary electrons. These can than be used to further amplify the signal in a cascade.
• Gain of 106-108.• Detectors “age” and require regular replacement.
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Detectors: MCP
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Microchannel Plates (MCP)
http://encyclopedia2.thefreedictionary.com/
Note: ion counting detectors also give signals of impact of energetic neutrals, electrons and photons. Therefore, care has to be taken, not to allow other particles than the mass-analysed ions to hit the detector.
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Tandem mass spectrometry
• MS/MS, MS2 or tandem mass spectrometry• Tandem-in-space can be performed on (ion beam-
transmitting) Hybrid instruments with 2 distinct mass analysers.
• Tandem-in-time in Ion traps: ion selection, activation and product ion analysis in the very same place.
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Product Ion Scan1. Precursor or “Parent”
Ions are selected and isolated
2. Fragmentation by CID
3. Product or “Daughter” Ions are characterised by Q3
4. Most common MS/MS experiment –used for protein ID
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Precursor Ion Scan1. Product Ion is
selected and Q3 is parked
2. Q1 scans normally3. Only precursors
which fragment to produce a selected product ion are detected.
4. Often used to target peptides with specific modifications resulting in “reporter ions”.
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Neutral Loss Scan1. The mass of a functional
group whose loss is to be detected is selected
2. Both Q1 and Q3 scan simultaneously, offset by the selected “neutral loss” mass
3. Fragmentation by CID4. Product ions are
detected only when specified loss occurs in Q2, indicating the presence of the moiety of interest.
5. Often used for peptide modifications such as phosphorylation.
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Selected Reaction Monitoring (SRM)
1. Precursor and product ion mass need to be known.
2. Q1 is fixed on specified mass.
3. Fragmentation by CID.4. Q3 is fixed on specified
fragment of mass selected in Q1.
5. Very commonly used for accurate peptide/small molecule quantitation.