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

The technique Offers 10-100 time higher mass resolution, resolving power and mass accuracy than any other mass analysis technique Orbitrap Balough 2004 (LC GC Europe)

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)

The set-up Basic set-up for all types of mass spectrometers www.premierbiosoft.com

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

Sample ionisation Electrospray Ionisation (ESI) www.lamondlab.com www.newobjective.com www.lamondlab.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 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…

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

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)

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

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 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 𝑚𝑑𝑒𝑓𝑒𝑐𝑡 = 𝑚𝑛𝑜𝑚𝑖𝑛𝑎𝑙 − 𝑚𝑒𝑥𝑎𝑐𝑡 Isotopic patterns in MS Charge state http://masspec.scripps.edu/

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

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

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 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

Location of the deamidated Asn49 residue could be shown applying ECD - http://www.bumc.bu.edu/ftms/research/isoaspartome/ Location of the deamidated Asn49 residue could be shown applying ECD

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

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!

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

Thank you!

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 =1.007825 Average mass: average atomic mass value www.wikipedia.org e.g. 1H and 2H (D) with R.A. of 100% and 0.0115% (H = 1.00794) Exact mass: theoretical mass Accurate mass: measured mass

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