1. 2th Student Symposium, IPHC Johann STOJKO 2013-05-24 Ion Mobility Mass Spectrometry (IMS): A promisingtool for biomoleculescharacterization Mail: johann.stojko@etu.unistra.fr Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO), Strasbourg

2. Summary I) Personalpresentation Who? Where? II) Introduction Somereminders: What? Basic definitions First steps in Mass Spectrometry world! III) Principles How? Native Mass Spectrometry, Ion Mobility (IMS) IV) Applications What for? Prospects

3. Personalpresentation LUXEMBOURG PARIS • Home region: Verdun, Lorraine • Education: Molecularbiology and biochemistry in Nancy, Lorraine STRASBOURG NANCY • Internship: • Auto-immunology in Luxembourg • Enzymology in Paris • Current position: PhDstudent in LSMBO, ECPM, Strasbourg • (ServierIndustryfellowship) Daltons? John Dalton (1766-1844) UNITED STATES OF AMERICA • British chemist and physicist • Studies on Daltonism • Meteorologicalresearch • Atomictheory (1808) • Atomic mass unity

4. Introduction • Biomolecules? «  Chemical compounds found in living organisms, mainlycomposed of carbon, hydrogen, oxygen, nitrogen, sulfur and phosphorus, and taking part in metabolicprocesses of theseorganisms »: Glucids, lipids, nucleicacids, aminoacids… • Protein? « Concatenation of aminoacidslinked by a peptide bond, essential for cellular functionwhereitcanplay a catalyticalrole » Combination & assembly Complexproteins 20 canonical aminoacids (Eucaryotes) • Ions? « Molecule or atomhavinglost or won one or severalelectrons, givingit an electric charge » • Acidaminoacids • Basic aminoacids + + 3 3 Proton release Proton capture + H Negative charge Positive charge - - Asp Glu Arg Lys His

5. Introduction M + z1mH M + z2 mH X1 = X2 = z1 z2 z2 = z1 - 1 • 2equations • 2 unknowns High sensitivity and resolution Destructive Mass determination of injected compounds How: m/z ratio measurement of ions Analytical technique Mass Spectrometry Positive or negative mode Gaseous phase X2 - 1 z1 = Biological support: Proteins and associated peptides Z measurement: X2 - X1 2 fields: Proteomics and supramolecularapproachs Denaturing conditions Native conditions Non covalent interactions studies (complexomics, interactomics, titration, affinity, stoichiometry, competition) Sequencing, identification characterisation & quantification of biomarkers 70% of labactivity 30% of labactivity

6. Principles of Mass Spectrometry: Workflow • Mass Spectrometry: Give informations about m/z ratios of ions Interpretation Gel separation Concentration LC coupling nanomate Preparation & pre-fractionation Sample introduction Data processing (bioinformatic system) needle syringe desalting Digestion digitizing Quadrupole MCP Ion detector TOF Mass analyser (combine them all!) Ion detection MALDI (solid) Electrospray (liquid) Orbitrap Soft ion source Ion trap m/z based ion separation Vaccumpumps Ion production

7. Native Mass Spectrometry: Someprecautions T°, pH T°, pH, [salts] Ionicstrenght Solventapolarity Denaturation Desalting Interactions loss Entireproteins native conformation maintaining Salt removing Non covalent interactions keeping Gas phase transfert & ionisation pusher detector 30 – 200µs + + + + + + 2+ (7501) 4+ (11251) 5+ 3+ Protonation of basic aminoacids (mode +) + Source + + 4+ + Ions focus and transmission + + 2+ + + 3+ (5001) 5+ (9001) 10 kDa + 5 kDa 15 kDa 20 kDa + 25 kDa 45 kDa • TOF: Arrival time conversion in m/z value • t when m and z , sowhen m/z Ions separation

8. Ion Mobility: Principle • TriWavetechnology (Synapt G2): Additionalseparative dimension! Ion gate TOF analyzer 5-40 ms IMS cell Ion storage separation transmission Separationparameters in ion mobility (K): • Potential impulsion (« wave n»): N2 20Å2 Surfing the electricpotentialwave 10Å2 CCS (Collison cross section Ω) 2+ K whenΩ « Roll over » effect 2+ Waitingfor the nextwave: separation! Charge state K when z 2+ 3+ 20kDa 10kDa Information: drift time! Size 2+ 2+ Lens number in IMS cell K when m Ion mobility or 1/dt

9. Applications m/z m/z Ion mobility intensity Native mass spectrum (2D) Driftscopeview (3D) 8+ Drift time 8+ 3000 8+ 9+ Intensity 8+ 2500 9+ 8+ 9+ 9+ 2000 9+ 8+ 9+ 1500 m/z Drift time (ms) • Mass measurement & intensity monitoring of species • Conformational data (X Ray, NMR, SAXS compatibility) • Complexomics & interactomics: • Proteomics: • Protein-protein & protein-ligand interactions screening • Additionalseparative dimension • Non covalent and covalent modifications studies • Coeluted peptides resolution • Titration experiments • Complexomics & interactomics: • Bindingaffinitymeasurements • Structural reorganizations & conformational changes • Stoichiometrydetermination • Isobaric and isomeric compounds separation • Bindingcompetitions • Kineticstudies • Kineticstudies

10. BDD Thankyou for your attention! LSMBO SCIENCES