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FT-ICR MS
An introduction and possible applications
Ariane Kahnt
Fourier transform ion cyclotron resonance mass spectrometry
The technique
• Offers 10-100 time higher mass resolution, resolving power and mass accuracy than any other mass analysis technique
Balough 2004 (LC GC Europe)
Orbitrap
• Hybrid instrument available in SBS: LTQ FT (i.e. ion trap - Fourier transform ion cyclotron resonance mass spectrometer from Thermo Scientific)
• Basic set-up for all types of mass spectrometers
The set-up
www.premierbiosoft.com
Sample introduction
• Hyphenation to liquid chromatography (LC) or direct infusion possible
LC
Sample ionisation
• Electrospray Ionisation (ESI)
www.lamondlab.com
www.newobjective.com
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
ESI
Q00
Q0 Q1
3 sets of octapoles
• Focussing and guiding of ions
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
• Ions can be stored, isolated and fragmented in the IT
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
Barrow et al., 2004 (The Analyst)
Ion cyclotron resonance analyser
Marshall and Hendrickson, 2008 (Annu. Rev. Anal. Chem.)
time-domain image-current signal
frequency spectrum mass spectrum
excited ion cyclotron rotation
Mass resolution versus resolving power
• Mass analyser must resolve adjacent peaks in complex samples
• Ability of an instrument to separate closely spaced peaks is called resolving power
• Resolution is calculated from the acquired data
• FWHM (full width at half maximum) definition
R
http://masspec.scripps.edu/Marshall and Hendrickson, 2008 (Annu. Rev. Anal. Chem.)
Some more things to consider…
• Mass defect– Characteristic for each atom – Arises from nuclear binding energy
• Charge state
𝑚𝑑𝑒𝑓𝑒𝑐𝑡❑=𝑚𝑛𝑜𝑚𝑖𝑛𝑎𝑙−𝑚𝑒𝑥 𝑎𝑐𝑡❑
• Isotopic patterns in MS
http://masspec.scripps.edu/
FT-ICR MS applications in biological sciences
• Protein characterisation
Thelen and Miernyk, 2012 (Biochem. J.)
• Metabolomics • Lipidomics
Intact protein characterization
• 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
• “Top-down” proteomics
• Tandem mass spectrometry
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
http://www.bumc.bu.edu/
Capability of electron capture dissociation (ECD)
• ECD involves capture of a thermal electron by the protonated peptide/protein
Kelleher, 2004 (Anal. Chem.)
• Causes peptide fragmentation from N-Ca bond
- Production of N-terminal c-type and C-terminal z-type ions
http://www.bumc.bu.edu/ftms/research/isoaspartome/
• Location of the deamidated Asn49 residue could be shown applying ECD
Soldi et al., 2013 (Int. J. Mol. Sci.)
• Combinational approach
Peptide and protein tagging methodologies
• Mass defect labelling for intact or digested proteins
Þ 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.)
• 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.”
But most importantly….
Roy Martin from Waters
Thank you!
• Nominal mass: integer mass value
• Average mass:
• Monoisotopic mass: exact mass of the most abundant isotopes of each element
average atomic mass valuee.g. 1H and 2H (D) with R.A. of 100% and 0.0115% (H = 1.00794)
e.g. H =1
e.g. H =1.007825
Chemical formula: C153H224N43O49S
www.wikipedia.org
• Accurate mass: measured mass
• Exact mass: theoretical mass
• Instrument calibration!
• Error of mass accuracy measurements
• Mass accuracy (in general) depends on several parameters :– Resolving power– S/N ratio of peaks, peak shape, overlapping species?– Scanning method, scan rate
Balough, 2004 (LC GC Europe)