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Lecture notes of 7 pages for the course Techniques For Biological And Chemical Sciences at QMUL
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Principles and applications of Mass SpectrometryLearning outcomes:
1. Explain the effects of:
- Multiple charges, Pseudo-molecular & Isotopes ions on the appearance of mass spectra.
2. Use mass spectra to determine molecular weight and confirm elemental composition
3. Explain how mass spectrometry can assist in the characterisation and structure
determination of proteins
Effect of multiple charge
The mass spectrum is then deconvoluted from the range of charges to provide the molecular
ion, which means all the multiple charged species are recalculated into its singly-charged
form and grouped together according to the m/z value and peak width.
Electrospray ionisation technique
- Soft ionisation technique (little fragmentation)
- Determine molecular weight for biological macromolecules
- Process does not fragment macromolecules into smaller charged particles
- Instead the macromolecule is being ionised into small droplets
- The small droplets are further desolvated (removing water between enzyme and
substrate for a chemical reaction to take place) into even smaller droplets, which creates
molecules with attached protons.
- These protonated and desolvated molecular ions will then be passed through the mass
analyser to the detector and the mass of the sample can be detected.
Mass spectrum of cytochrome C
Determination of molecular weight
Electrospray spectra of large molecules, such as proteins typically show series of ions of
general formula (M + zH+)
- Z is an integer,1,2,3,4…., which increases one unit at a time
- The first few numbers of the series (z=1,2,3,4,…) are often not observable but the data
may be ‘deconvoluted’ to provide the mass of the molecular ion
, Formation of multiple charged ions means that v.high molecular substances can still give
manageable m/z values
E.g- cytochrome C has a relative molecular mass of 12,384 Da but forms and ion with a 10+
charge and a mass of 12,394 Da by addition of 10 protons, giving m/z approximately 12,394.
Effect of pseudo-molecular ions
Pseudo-molecular ions are ions formed by soft ionisation technique in which the molecular
ion may be present with a proton or other cations attached or, for negative ions, with a
proton removed
The transfer of protons from the matrix to the analyte and the production of
pseudomolecular, sometimes with multiple charges.
- If Na+ salt is present then [M+Na]+ ions may be formed.
‘soft’ ionisation techniques such as electrospray, give pseudomolecular ions such as M+H+,
M+NH4+ and M+Na+.
Pre-treatment to sample to desalt protein sample important
Effect of isotopes
Masses of common isotopes
Example small molecule mass specrum showng isotope pattern for [M+H+] ion
For a molecule with one carbon in it 99% 12C and 1% 13C
With ten Carbons in molecule
90% of the molecules will have all ten Carbons as 12C
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