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)