Mass Spectrometry Flashcards
Mass Spectrometer
Instrument used to define covalent structures of substances by ionising, separating and detecting molecular and fragment ions according to their mass to charge ratios
Benefits of MS
- study complex and crude mixtures
2. sensitive (don’t need large starting volumes)
Parts of a Spectrometer
- ion source: converts sample to gas phase ions
- mass analyser: separates based on m/z of ion
- detector: detects abundance
Spectrometry Graph
- intensity vs. m/z ratio
- most abundant species given a value of 100% and all other components are expressed as relative % of this species
Ionisation Methods
- Electron Impact (hard): 1-1000 Da
- Electrospray Ionisation (soft): greater than 500,000
- Matrix Assisted Laser Desorption Ionisation (soft): up to 500,000
Electron Impact Ionisation
- 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
MALDI
- 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
MALDI Lasers
- nitrogen gas UV laser
2. solid state UV laser: high repetition rate
MALDI Matrices
- 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
Delayed Extraction
- 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
Electrospray Ionisation
- 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)
Nano-ES
- 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
MS Analyzers
- quadrupole
- time of flight
- ion trap
- orbitrap
Factors of a Mass Analyzer
- 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
Quadrupole
- 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
Quadrupole Mass Filters
- 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
Ion Trap
- 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
Orbitrap Analyzer
- 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
Orbitrap Characteristics
- highest performing
- high resolution
- high mass accuracy
- good upper mass range/ion transmission
TOF analyser
- 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
Reflectrons
- 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
Ion Detector Types
- PM-photomultiplier: detects photons
- EM-electron multiplier: detects electrons
- MCP: microchannel plate array detectors for multiple m/z values simultaneously
PM Detector
- ions strike a dynode causing electron emission
- electrons strike phosphorous screen releasing photon burst
- photons multiplied for amplification of sensitivity
EM Detector
- 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
MCP Detectors
- many separate channels for increased spatial resolution
- multiple electron multipliers arrayed
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
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
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
Types of Molecular Ions
- 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
- MALDI: singly charged cations and anions (take into account counterions)
- Electrospray: multiply charged cations and anions; this increased charged decreases the m/z ratio
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
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
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
Triple Quadrupole
- Q1: mass filter selects ions of interest
- Q2: collision chamber generating ions
- Q3: mass analyser separates/detects
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
MALDI TOF-TOF
- MALDI ion source with two TOF detectors and a reflectron
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)
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
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
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
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
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)
A ion Fragmentation
- secondary fragmentation of B ion
- loss of charged carboxyl group to give alpha ion
- act as confirmation of B ion presence