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Protein Science > Mass Spectrometry > Flashcards

Mass Spectrometry Flashcards

1
Q

Mass Spectrometer

A

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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2
Q

Benefits of MS

A
  1. study complex and crude mixtures

2. sensitive (don’t need large starting volumes)

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3
Q

Parts of a Spectrometer

A
  1. ion source: converts sample to gas phase ions
  2. mass analyser: separates based on m/z of ion
  3. detector: detects abundance
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4
Q

Spectrometry Graph

A
  • 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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5
Q

Ionisation Methods

A
  1. Electron Impact (hard): 1-1000 Da
  2. Electrospray Ionisation (soft): greater than 500,000
  3. Matrix Assisted Laser Desorption Ionisation (soft): up to 500,000
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6
Q

Electron Impact Ionisation

A
  • 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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7
Q

MALDI

A
  • 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
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8
Q

MALDI Lasers

A
  1. nitrogen gas UV laser

2. solid state UV laser: high repetition rate

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9
Q

MALDI Matrices

A
  • 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
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10
Q

Delayed Extraction

A
  • 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
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11
Q

Electrospray Ionisation

A
  • 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)
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12
Q

Nano-ES

A
  • 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
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13
Q

MS Analyzers

A
  1. quadrupole
  2. time of flight
  3. ion trap
  4. orbitrap
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14
Q

Factors of a Mass Analyzer

A
  1. upper mass limit: largest m/z ion they can separate
  2. ion transmission: how many produced ions can be separated to reach the detector
  3. resolution: how good is it at separating similar m/z ions
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15
Q

Quadrupole

A
  • 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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16
Q

Quadrupole Mass Filters

A
  • 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
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17
Q

Ion Trap

A
  • 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
18
Q

Orbitrap Analyzer

A
  • 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

A
  • highest performing
  • high resolution
  • high mass accuracy
  • good upper mass range/ion transmission
20
Q

TOF analyser

A
  • 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
21
Q

Reflectrons

A
  • 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
22
Q

Ion Detector Types

A
  1. PM-photomultiplier: detects photons
  2. EM-electron multiplier: detects electrons
  3. MCP: microchannel plate array detectors for multiple m/z values simultaneously
23
Q

PM Detector

A
  • ions strike a dynode causing electron emission
  • electrons strike phosphorous screen releasing photon burst
  • photons multiplied for amplification of sensitivity
24
Q

EM Detector

A
  • 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
25
Q

MCP Detectors

A
  • many separate channels for increased spatial resolution

- multiple electron multipliers arrayed

26
Q

Types of Instruments

A
  • MALDI-TOF common for mass fingerprinting complex polymer mixtures and larger molecule analysis
  • ES comon with quadrupole, ion trap, orbitrap or Q-TOF instruments
27
Q

Hybrid Instruments

A
  • 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
28
Q

Fragmentation

A
  • 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
29
Q

Types of Molecular Ions

A
  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
30
Q

Pseudo-ions

A
  • 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
31
Q

EI-MS Fragment Ions

A
  • 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
32
Q

Collisional Activation

A
  • 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
33
Q

Triple Quadrupole

A
  • Q1: mass filter selects ions of interest
  • Q2: collision chamber generating ions
  • Q3: mass analyser separates/detects
34
Q

Q-TOF

A
  • 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
35
Q

MALDI TOF-TOF

A
  • MALDI ion source with two TOF detectors and a reflectron
36
Q

ES-MS/MS Peptide Sequencing

A
  • 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)
37
Q

Recognising multiply charged ions

A
  • 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
38
Q

Peptide Fragmentation

A
  • 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
39
Q

B ion fragmentation - N terminal

A
  • 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
40
Q

Y ion fragmentation - C terminal

A
  • 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
41
Q

Fragment Ion Masses

A

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)

42
Q

A ion Fragmentation

A
  • secondary fragmentation of B ion
  • loss of charged carboxyl group to give alpha ion
  • act as confirmation of B ion presence

Protein Science (11 decks)

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