Mass Spectrometry Flashcards
1
Q
Mass Spectrometer
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Instrument used to define covalent structures of substances by ionising, separating and detecting molecular and fragment ions according to their mass to charge ratios
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Q
Benefits of MS
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- study complex and crude mixtures
2. sensitive (don’t need large starting volumes)
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Q
Parts of a Spectrometer
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- ion source: converts sample to gas phase ions
- mass analyser: separates based on m/z of ion
- detector: detects abundance
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Spectrometry Graph
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- intensity vs. m/z ratio
- most abundant species given a value of 100% and all other components are expressed as relative % of this species
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Q
Ionisation Methods
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- Electron Impact (hard): 1-1000 Da
- Electrospray Ionisation (soft): greater than 500,000
- Matrix Assisted Laser Desorption Ionisation (soft): up to 500,000
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Q
Electron Impact Ionisation
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- there is a size limit because the sample must already be in the gas phase
- sample introduced into source by heating until evaporation
- there is no direct collision; the gas phase sample is bombarded with electrons from a filament
- the beam comes into proximity and repels an electron from the outer orbital that becomes a radical cation
- there is an excess of energy allowing fragmentation of the molecular ions
- magnets focus the beam to increase chances of ionization
- a charged ion repeller controls the positively charged ions to push them into the MS
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Q
MALDI
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- ionisation from solid phase
- sample mixed with liquid matrix and applied to the metal target where it drys out and becomes crystalline
- sample embedded in a low Mw crystalline matrix with an absorption maximum near the wavelength of the laser used for ionisation
- matrix absorbs the laser pulse and energy is transferred to the sample for ionisation
- similar to ‘flash evaporation’
- the metal target can be charged to repulse ions of a charge into the MS
8
Q
MALDI Lasers
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- nitrogen gas UV laser
2. solid state UV laser: high repetition rate
9
Q
MALDI Matrices
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- allows initial energy absorption, conversion, and transfer
- need absorption max. similar to emission max of laser
- small organic molecules with aromatic groups or double/triple bonds act as chromophores to allow energy absorption from the laser
10
Q
Delayed Extraction
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- the pulse of ions is kept in the source for a short time after the pulse to allow ions formed deep in the matrix to emerge and catch up with surface ions
- ions of the same mass from different places in the source have different speeds
- this uneven energy distribution creates a broader low quality spectra
- application of a field accelerates the slower ions closer to the pulse more so they catch up
11
Q
Electrospray Ionisation
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- atmospheric pressure ionisation
- sample introduced into source via a narrow glass capillary coated in gold
- a high voltage is applied to the tip and the sample emerging is dispersed as aerosol of highly charged droplets
- select for specific charge by controlling electrical potential on plates surrounding the ions
- a drying gas flows around the outside
- as solvent evaporates the droplets become unstable due to a high surface charge where like ions come into close proximity
- eventually you get naked ions whose charge corresponds to the charge of the original molecule (depends on pH and electrical potential)
12
Q
Nano-ES
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- much more sensitive than ES as it needs much less starting material with a higher flow rate
- time for many experiments on a single sample
- can link to chromatography to analyse complex mixtures
13
Q
MS Analyzers
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- quadrupole
- time of flight
- ion trap
- orbitrap
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Q
Factors of a Mass Analyzer
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- upper mass limit: largest m/z ion they can separate
- ion transmission: how many produced ions can be separated to reach the detector
- resolution: how good is it at separating similar m/z ions
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Q
Quadrupole
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- quadrupolar electric field to separate ions consisting of 4 parallel rods
- each diagonal pair of rods connected electrically
- field obtained by application of a voltage made of a DC component and RF potential
- some ions of certain m/z are in harmony with the field and fly through
- those out of harmony are attracted to a pole and are destroyed
- can ‘scan’ through different m/z ranges and mke calibration curve
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Q
Quadrupole Mass Filters
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- m/z ratios of 4,000 observed (low)
- low resolution
- small, cheap, robust
- rapid scanning through a m/z range
- low sensitivity as not all ions are detected
17
Q
Ion Trap
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- stores ions using fields generated by RF and DC voltages applied to electrodes arranged in a sandwich geometry
- sandwich composed of ring electrode in the middle with cap electrodes on one end
- 3D quadrupole field trapping ions in space
- increasing strength of the field rotates ions with more energy and a bigger radius
- at different strengths ions of a m/z are ejected to the detector
- linear traps have a larger volume for better ion transmission and resolution
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Q
Orbitrap Analyzer
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- ions trapped by static electrostatic field
- ions orbit around central electrode and oscillate in axial direction around barrel electrode
- m/z ratio relates to the frequency of ion oscillation along the axial direction
- FT converts time-domain signal to m/z
19
Q
Orbitrap Characteristics
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- highest performing
- high resolution
- high mass accuracy
- good upper mass range/ion transmission
20
Q
TOF analyser
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- ions separated by differences in velocities as they move in a straight path to the detector
- larger m/z ions are slower and take longer to traverse
- generate calibration curve relating m/z to time taken
- unlimited mass range
- can detect vast majority of ions
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Q
Reflectrons
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- early TOF analyzers were poor accurate
- new ones use reflectrons to correct for the effect of KE distribution of the ions and give a longer flight path (correct for slight energy changes of same m/z ions)
- reflection is an ion mirror that reverses the direction of travel of the ions - ions of greater KE penetrate further and therefore have a longer flight path
- this corrects the energy imbalance as the faster ion will turn around later and then be delayed to catch up to the slower ion
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Q
Ion Detector Types
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- PM-photomultiplier: detects photons
- EM-electron multiplier: detects electrons
- MCP: microchannel plate array detectors for multiple m/z values simultaneously
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Q
PM Detector
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- ions strike a dynode causing electron emission
- electrons strike phosphorous screen releasing photon burst
- photons multiplied for amplification of sensitivity
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Q
EM Detector
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- ions attracted to a electron multiplier
- strike a coated surface to release secondary electrons
- hit surface surface again to release more secondary electrons
- cascade to produce electrical signal
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MCP Detectors
- many separate channels for increased spatial resolution
| - multiple electron multipliers arrayed
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Types of Instruments
- MALDI-TOF common for mass fingerprinting complex polymer mixtures and larger molecule analysis
- ES comon with quadrupole, ion trap, orbitrap or Q-TOF instruments
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Hybrid Instruments
- two or more analyzers in tandem
- used in MS/MS experiments for 2D analysis
- high specificity and sensitivity
eg: MALDI ion source with ion trap and TOF with reflectron
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Fragmentation
- certain amount of energy needed to generate the molecular ion
- if you have an excess of energy you then generated fragment ions
- fragment ions are used to study the structure of peptides
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Types of Molecular Ions
1. Electron Impact: radical cations; the loss of a single electron of negligible mass means the m/z ratio is equal to the mass of the species
2. MALDI: singly charged cations and anions (take into account counterions)
3. Electrospray: multiply charged cations and anions; this increased charged decreases the m/z ratio
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Pseudo-ions
- soft ionisation sources produce pseudo molecular ions
- in order to obtain a charged species we use a counter ion associated with it
- an ion can be protonated, deprotonated, sodiated, or potiassiated
- the mass of the counter ion needs to be taken into account when calculating the m/z
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EI-MS Fragment Ions
- spectra doesn't contain predicted molecular ion
- hard technique with excess energy fragmenting the MI
- lower m/z signals are these fragment ions derived from molecular ion
* doesn't mean this always occurs*
- alkyloid species with conjugate ring system can absorb excess energy and not fragment so often
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Collisional Activation
- used in soft techniques like MALDI or ES
- two or more analyzers connected in tandem
- ions are selected by first analyzer and are collisionally activated as they pass through a collision chamber located between the two analyzers
- fragment ions plus any non fragmented molecular ions are separated by the second analyser and detected
- the gas is insert and the collision transfers enough KE to induce fragmentation
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Triple Quadrupole
- Q1: mass filter selects ions of interest
- Q2: collision chamber generating ions
- Q3: mass analyser separates/detects
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Q-TOF
- high resolution/sensitivity
- MS or MS/MS mode
- MS mode: quadrupole allows all ions through and TOF is mass analyzer. pulses accelerate ions into the TOF to record spectrum
- MS/MS mode: quadrupole only allows select ions into collision cell with gas TOF is again the mass analyzer
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MALDI TOF-TOF
- MALDI ion source with two TOF detectors and a reflectron
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ES-MS/MS Peptide Sequencing
- ES generates multiply charged ions that are easier to fragment than singly charged peptide molecular ions
- these ions require less collisional energy than singly charged ions & majority of impurity derived signals are singly charged
- peptide derived fragment ions will predominate in the MS/MS data even if singly charged contaminants are present at the same m/z value as the peptide MI
- MCI recognised by interval between isotope peaks
- trypsin is enzyme used to digest proteins as typtic peptides have a minimum of two charges (N terminus plus C terminal K/R)
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Recognising multiply charged ions
- use the interval between each peak in the cluster (interval between carbon-12 and carbon-13)
- allows identification of charge state
- 1% of all peptide carbons will be carbon-13 forming an isotope cluster for each peptide MI
- singly charged: separation is 1
- double charged: separation is 0.5
- triply charged: separation is 0.33
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Peptide Fragmentation
- nonrandom pathways of fragmentation
- proton is transferred to the peptide bond where cleavage takes place
- peptide sequencing works due to the lack of symmetry on the N and C terminal ends
- because the ends of the peptides differ in groups the masses differ : this asymmetry is fundamental in interpretation
- L/I cannot de distinguished
- MALDI-TOF TOF favors N-G cleavage due to chemistry
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B ion fragmentation - N terminal
- start at N terminus and work to C terminus
- ionising proton attaches to N of peptide bond and peptide bond breaks
- carboxyl group becomes charged with triple bond between oxygen/carbon (b ion)
- neutral species formed
- generated from many positions giving combinations of different ions
- can determine Mw of each residue in order
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Y ion fragmentation - C terminal
- start at C terminus and work to N terminus
- produce quaternary charged N
- ionising proton attaches to N of peptide bond and hydrogen on alpha carbon attaches to N to form quaternary charge
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Fragment Ion Masses
Mass of b-ions = Σ (residue masses) + 1 (H+)
Mass of y-ions = Σ (residue masses) + 19 (H2O+H+)
Mass of a-ions = mass of b-ions – 28 (CO)
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A ion Fragmentation
- secondary fragmentation of B ion
- loss of charged carboxyl group to give alpha ion
- act as confirmation of B ion presence
