04_mass-spectronomy

1898: Deflection of ions by electric and magnetic fields

> 1898: Determination of masses and isotope ratios of chemical elements

1943: first commercial mass spectrometer

> 1980: Development of soft ionisation methods for biomolecules (no denaturation of proteins)

chemical elements determined by number of protons (governed by electron cloud)

istopes of that element determined by number of neutrons

mass definitions

• nominal/integer mass: convenient w. o. physical meaning, not measured

• chemical/average mass: sum of all stable isotopes, found on labels, measureable

• monoisotopic mass: sum of most abundant stable isotopes, lightest for most isotopes, measureable

  -> natural distribution cause of different stable isotopes and natural abundances (C^12, C^13, etc.)

  -> different combinations of different stable isotopes for a given sequence possible

  => the higher the resultion, the more details from/for different isotopes

  
  

1. Ion source from sample

2. Mass analyzer

3. Detector

• manipulation of ions by electric and magnetic fields

• mass to charge ratio (m/z) measured by mass spectrometers

  • no. of charges determined by isotope spacing (btw. two isotopes +/- 1 aka the mass of a neutron)

  • calculation of charge state deconvolution

    1. m/z (monoisotopic peak)

    2. determination of z (isotope spacing)

    3. m = m/z * z

    4. neutral peptide mass = m-n*(no. of proton mass)

• Electrospray Ionisation (ESI)

• Matrix-assisted laser desorption Ionisation (MALDI)

-> charging ions of sample

matrix assisted laser desorption ionisation from a solid state in a vacuum

-> costs energy from environment

1. matrix assisted: formation of crystals with to analyse molecules inside

2. laser: absorbed by UV-active matrix

   -> matching laser with molecules

   -> energy difference co-incides w. photon -> emission

3. Desorption: Flash evaporation, sublimation w. o. liquid phase

4. soft Ionisation: Protein transfer, photodecomposition

-> production of single charged ions by transfer of protons (change of spectrometer configurations to decide for different donors)

=> signal indication no indication for absolute peptide quantitiy due to fly abilitys (in vacuum) based on composition of AAs

=>

=> further analysis and detection by mass analyzers and detectors

electrospray ionisation from liquid state

-> direct coupling of LC and MS

1. applying charge to the outlet of a capillary atomizing particles into tiny charged droplets (electrostatic force > surface tension -> Taylor cone production)

2. droplet fission: breaking down droplets into smaller drops

3. ionisation

   • Ion evaporation model (IEM, active process): small radii drops w. high surface charge densities and strong electrostatic fields -> field desorption of solvated analyte ions from the surface of the droplets

   • Charge residue model (CRM, passive process): no more droplet and no more solvent -> only charge remains

-> multiply charged

-> signal intensity depending on analyte concentration

-> the smaller the liquid volume and/or flow rate, the better the response

• Time-of-flight (TOF)

• Quadrupole

• Ion trap

• Orbitrap

• Properties

Time-of-flight mass analyzer

analysing time of flight of analyte ions traveling through a flight tube at the same voltage

-> speed depending on mass/charge of analyte ion

=> the higher the mass, the slower the ion

—> kinetic energy determined by no. of charges

—> m/z relative to velocity and thus drift time

==> acceleration w. same kinetic energy -> drifting -> detection (of those who managed to drift to the “destination)

—-> for same type different energy possible through some way of dispersion

Quadrupole mass analyzer

isolation of a single ion and analyzation of charge and mass

spiral through arrangement w. overlay of two fields (1 constant, 1 alternating) -> only particular ions of particular m/z w. stable oscillation -> passing of those ions and safely transmitted -> oscillation btw. electric quadrupole w. alternating voltage

=> increase of power/amplitude throughout to get bigger ions increasingly

—> limitations of m/z range (typically 4 - 8,000 m/z)

IT - ion trap mass analyser

3D

stable oscillating trajectory in a quadrupolqr field

-> m/z dependent detection by scanning potentials to destabilise the ions motions of particular m/z value and eject them through exit endcap

—> storing ions of interest

=> limited m/z range

2D/linear

trapping by scanning potentials to destabilise ion motions of particular m/z values and eject them through rods

—> storing ions of interest

=> limited m/z range

==> advantage to 3 D: storing ability of many more ions

OT - orbitrap mass analyzer

outer electrode + central spindle

-> oscilation of ions around stable spindel and 3D movements of ions within orbitrap

Lorentz law: moving charges induce curent -> recording of oscillation frequency around z-axis to detect ions w. mz

=> complex-domain signal by many ions w. different m/z at same time => Fourier Transformation to deconvolute

• precision: reproduction of measurement

• accuracy: closeness to true value

-> measured in ppm (parts per million)

• resolution = m/z / width

  • peak width = full-width-at-half-maximum

  • ability to seperate two adjacent masses/isotopes

-> accuracy and precision increase with increasing resolution of mass measurement

destructive

generation of secondary electrons through ion impact

non-destructive

generation of an image current through ion movement

-> passing between two metal surfaces with weak currents while oscillating

MALDI-TOF

• rapid analysis + relatively cheap

• no longer common

• analysis of synthetic peptides or oligonucleotides

• MALDI biotyper

ESI-QUAD / ESI-TRAP / ESI-Orbi

Q-TOF

Ion trap-orbitrap