1. An introduction and possible applications Ariane Kahnt
FT-ICR MS
Fourier transform ion cyclotron resonance mass spectrometry
An introduction and possible applications
Ariane Kahnt

2. The technique
Offers time higher mass resolution, resolving power and mass accuracy than any other mass analysis technique
Orbitrap
Balough 2004 (LC GC Europe)

3. Hybrid instrument available in SBS: LTQ FT (i. e
Hybrid instrument available in SBS: LTQ FT (i.e. ion trap - Fourier transform ion cyclotron resonance mass spectrometer from Thermo Scientific)

4. The set-up Basic set-up for all types of mass spectrometers

5. Sample introduction LC
Hyphenation to liquid chromatography (LC) or direct infusion possible LC

6. Sample ionisation Electrospray Ionisation (ESI) www.lamondlab.com

7. Ion transfer optics
After the introduction and formation of gas-phase ions, charged analytes are transferred by various octapole and quadrupole lenses to the mass analyser
Focussing and guiding of ions
Q00 Q0 Q1
3 sets of octapoles
ESI
From ESI via ion transfer capillary to tube lense (which focusses the charged species) and skimmer (that removes neutrals) to Q00 – lense 0 – Q0 lense L1 plus gate lense Q1 etc…

8. Ion trap
The linear ion trap is a fully operational MS detector but is at the same time also an ion preparation and injection system for the ion cyclotron MS
ESI
Linear ion traps have greater ion storage capacity than conventional 3D ion traps
Ion handling, selection, excitation capabilities of the ion trap can be used for the ion preparation prior ICR analysis
E.g. storage and ejection of all ions, or storage of only selected m/z or mass ranges, ion isolation
Ions can be stored, isolated and fragmented in the IT

9. Ion cyclotron resonance analyser
ICR cell is located in the centre of the magnetic field and traps gas phase ions in the centre using electric and magnetic fields
Excitation of ions to a larger cyclotron radius
Trapping electrodes, excitation electrodes and detection electrodes
Excitation of ions by the application of a radio frequency voltage (to their resonance cyclotron frequency)
Barrow et al., 2004 (The Analyst)

10. Ion cyclotron resonance analyser
excited ion cyclotron rotation
Marshall and Hendrickson, 2008 (Annu. Rev. Anal. Chem.)
time-domain image-current signal
frequency spectrum
mass spectrum

12. Mass analyser must resolve adjacent peaks in complex samples
Mass resolution versus resolving power
Ability of an instrument to separate closely spaced peaks is called resolving power
Resolution is calculated from the acquired data
R = 𝑚2 𝑚2−𝑚1 50%
FWHM (full width at half maximum) definition
Marshall and Hendrickson, 2008 (Annu. Rev. Anal. Chem.)

13. Some more things to consider…
Mass defect
Characteristic for each atom
Arises from nuclear binding energy
𝑚𝑑𝑒𝑓𝑒𝑐𝑡 = 𝑚𝑛𝑜𝑚𝑖𝑛𝑎𝑙 − 𝑚𝑒𝑥𝑎𝑐𝑡
Isotopic patterns in MS
Charge state

14. FT-ICR MS applications in biological sciences
Protein characterisation
Thelen and Miernyk, 2012 (Biochem. J.)
Metabolomics
Lipidomics

15. Intact protein characterization
“Top-down” proteomics
Non-routine analysis for proteins >50 kDa
Usually performed on single proteins or modest mixtures
Can help in measuring variations of coding polymorphisms, alternative splicing, diverse post-translational modifications
e.g. applied after classical (1D or 2D) gel electrophoresis
Top-down method provides the more accurate and complete information (protein size, quantification, precise characterization of PTMs, splicing forms) – has better sequence coverage (in comparison to bottom up method)
Tandem mass spectrometry

16. Protein isoforms and posttranslational modifications
E.g. non-enzymatic deamidation of asparagine (and to a lesser extent glutamine) in-vivo and in-vitro have important biological effects
Such as on enzyme activity, folding, proteolytic degradation

17. Capability of electron capture dissociation (ECD)
ECD involves capture of a thermal electron by the protonated peptide/protein
Causes peptide fragmentation from N-Ca bond
Production of N-terminal c-fragments and C-terminal z-type fragments
Fragmentation happens before E-transfer within the molecule – therefore labile modification groups will stay intact!
Kelleher, 2004 (Anal. Chem.)
Production of N-terminal c-type and C-terminal z-type ions

18. Location of the deamidated Asn49 residue could be shown applying ECD-
Location of the deamidated Asn49 residue could be shown applying ECD

19. Combinational
approach
Soldi et al., 2013 (Int. J. Mol. Sci.)

20. Peptide and protein tagging methodologies
Mass defect labelling for intact or digested proteins
H is the dominant element responsible for the mass defect of a protein - BUT peptides cluster tightly in each individual mass unit
Incorporation of mass defect labels to alter the mass defect and to obtain a shift in mass plus using specific isotope pattern of labels
Hernandez et al., 2006 (Anal. Chem.)
Large overlap within these clusters; unambiguous ID of peptide based on accurate mass alone is impossible!
Mass shift decrease possible interferences and can enhance the number of identified proteins!

21. But most importantly…. A well prepared sample,
well-defined analytical goal,
appropriate use of accurate mass,
reproducible retention times and
good instrument control generates unassailable data!
“Make it run in triplicate, and [you] get real data from which to draw conclusions.”
Roy Martin from Waters

22. Thank you!

23. Nominal mass: integer mass value e.g. H =1
Monoisotopic mass: exact mass of the most abundant isotopes of each element
Chemical formula: C153H224N43O49S
e.g. H =
Average mass:
average atomic mass value
e.g. 1H and 2H (D) with R.A. of 100% and %
(H = )
Exact mass: theoretical mass
Accurate mass: measured mass

24. Instrument calibration!
Error of mass accuracy measurements
Balough, 2004 (LC GC Europe)
Mass accuracy (in general) depends on several parameters :
Resolving power
S/N ratio of peaks, peak shape, overlapping species?
Scanning method, scan rate