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Experimental methods for the spectroscopic study of ionic biomolecules in the gas phase

College of Photonics and Applications 7-11 August 2006 Hanoi. Experimental methods for the spectroscopic study of ionic biomolecules in the gas phase. Pierre Çarçabal Laboratoire de PhotoPhysique Moléculaire CNRS – Orsay – France pierre.carcabal@ppm.u-psud.fr. Layout. Introduction

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Experimental methods for the spectroscopic study of ionic biomolecules in the gas phase

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  1. College of Photonics and Applications 7-11 August 2006 Hanoi Experimental methods for the spectroscopic study of ionic biomolecules in the gas phase Pierre Çarçabal Laboratoire de PhotoPhysique Moléculaire CNRS – Orsay – France pierre.carcabal@ppm.u-psud.fr

  2. Layout • Introduction • WHY? WHY? WHY? • Common ions production methods • Electrospray • Maldi • Cooling methods • Why cooling? • Supersonic expansions • Collisional cooling • Droplet evaporative cooling • Superfluid Helium Nanodroplet cooling • COUPLING A MALDI SOURCE TO A SUPERSONIC EXPANSION • Basic spectroscopy techniques applied toions • Advantages and drawbacks of working with ions (cf François Piuzzi lecture on spectroscopy of neutral species) • Examples of applications • IRMPD using Free Electron Lasers • Photodissociation electronic spectroscopy of protonated ions

  3. Layout • Introduction • WHY? WHY? WHY? • Common ions production methods • Electrospray • Maldi • Cooling methods • Why cooling? • Supersonic expansions • Collisional cooling • Droplet evaporative cooling • Superfluid Helium Nanodroplet cooling • COUPLING A MALDI SOURCE TO A SUPERSONIC EXPANSION • Basic spectroscopy techniques applied toions • Advantages and drawbacks of working with ions (cf François Piuzzi lecture on spectroscopy of neutral species) • Examples of applications • IRMPD using Free Electron Lasers • Photodissociation electronic spectroscopy of protonated ions

  4. Why bringing biomolecules intothe gas phase? • The biological medium is very complex • Molecular crowding • - WHO DOES WHAT? • - DOES IT DO IT BY ITSELF? • - CAN THE ENVIRONMENT AFFECT • MOLECULE’S ROLE? • - WHAT CAN GO WRONG? • MOTOR - LIVING SYSTEM • Gears - Isolated molecule • WHAT ARE THE MINIMUM • REQUIREMENTS TO DO THE JOB?

  5. Why bringing biomolecules intothe gas phase? • If we want to understand in details how the whole thing (car) work weneed to understand its components(car > motor > gears) • The best environment to study the intrinsic properties of a small molecular system is in vaccuo (no interactions, no collisions)

  6. What can gas phase spectroscopy tell? • Hydrogen bonding plays a central rolein ALL biological processes • What is the H bonding? • Non covalent interaction between polar groups in a molecular assembly • charge/dipole-dipole (electrostatic interaction) • dipole-induced dipole (induction or polarisation interaction) • dispersion interaction • steric hindrance • repulsion

  7. What can gas phase spectroscopy tell? • Hydrogen bonding plays a central rolein ALL biological processes • What is the H bonding? • Non covalent interaction between polar groups in a molecular assembly • charge/dipole-dipole (electrostatic interaction) O H H d- O N H d+ d+ C O d- d+ d- d+ d- H O d+ d- MOLECULAR ASSEMBLY

  8. What can gas phase spectroscopy tell? • Hydrogen bonding plays a central role in ALL biological processes • What is the H bonding? MOLECULAR ASSEMBLY - single molecule → INTRAmolecular H bond - several molecules → INTERmolecular H bond O H H d- O N H d+ d+ C O d- d+ d- d+ d- H O d+ d- MOLECULAR ASSEMBLY

  9. What can gas phase spectroscopy tell? • Hydrogen bonding plays a central role in ALL biological processes • What is the H bonding? • What do H bonds do? • INTRAmolecular: structures stabilisation • INTERmolecular: promotes interaction • molecular recognition • adaptation to environment

  10. “Flexibility and Function. Upon binding iron, the protein lactoferrin undergoes conformational changes that allow other molecules to distinguish between the iron-free and the iron-bound forms.” From “Biochemistry”, J.M. Berg, J.L. Tymoczko, L. Stryer, Ed. Freeman, NY

  11. O H O H molecule What can gas phase spectroscopy tell? • INFRARED SPECTROSCOPY IS A VERY SENSITIVE PROBE OF • MOLECULAR STRUCTURE • HYDROGEN BONDING NH stretching COH bending OH stretching IR energy H O O H molecule x IR energy

  12. Example: Phenyl-a-D-mannopyranoside (apMan) DOUBLE RESONANCE IR-UV cG-g+ 0 ccG-g+ 3.2 cTt 3.7

  13. Why studying ions? • BIOMOLECULES ARE OFTEN IN THEIR IONIC STATES IN THE BIOLOGIC MEDIUM From “Biochemistry”, J.M. Berg, J.L. Tymoczko, L. Stryer, Ed. Freeman, NY

  14. Layout  • Introduction • WHY? WHY? WHY? WHAT? WHAT? WHAT? • Common ions production methods • Electrospray • Maldi • Cooling methods • Why cooling? • Supersonic expansions • Collisional cooling • Droplet evaporative cooling • Superfluid Helium Nanodroplet cooling • COUPLING A MALDI SOURCE TO A SUPERSONIC EXPANSION • Basic spectroscopy techniques applied toions • Advantages and drawbacks of working with ions (cf François Piuzzi lecture on spectroscopy of neutral species) • Examples of applications • IRMPD using Free Electron Lasers • Photodissociation electronic spectroscopy of protonated ions

  15. Ions production methods • What do we want: • to bring the molecules into the gas phase with as little fragmentation as possible • to produce a large enough amount of molecules in the desired ionic state • Protonated • Deprotonated • ZWITERIONIC (!!!!!)

  16. Ions production methods • TWO MAIN METHODS: • ELECTRO SPRAY IONISATION • MALDI

  17. Mulitcharged droplet Coulombic explosion + + + + + + + - - - + + - + + + + + + + + + + + - + + + + + + + + + + + + + + + + - + + + - + + + + + + - + + + - + + + + + + - - - - Isolated ions Solvent evaporation Electro Spray Ionization N2 Analyte + solvent - - analyser - 3 – 6 kV

  18. Electro Spray Ionization • It is now the most widely used method bythe mass spectrometry community • Very easy to use • Can produce highly charged ions • Use VERY little amount of analyte • But it has an important drawback for spectroscopists: it is a continuous source

  19. MALDI • Matrix Assisted Laser Desorption Ionization Laser N2 (337 nm) Nd:Yag (355 nm) Nd:Yag (266nm) Excimer CO2 Pusher Matrix ions Analyte ions Analyser

  20. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • Good absorption of the laser photons • Can co-crystallize with the analyte • Soluble in same solvent as the analyte • Good proton transfer • Protects the analyte

  21. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE?

  22. MALDI SOME COMMON MALDIMATRICES:

  23. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE? • HOW TO CHOOSE A MATRIX? • Depends on the laser to be used • Depends on the kind of molecule to be studied

  24. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE? • HOW TO CHOOSE A MATRIX? • HOW DOES IT WORK? • The SIMPLE explanation

  25. MALDI Laser Sample plate hn • 1. Sample (A) is mixed with excess matrix (M) and dried • on a MALDI plate. • 2. Laser pluse ionizes matrix molecules. • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. AH+ Variable Ground Grid Grid +20 kV

  26. Laser Sample plate hn • 1. Sample (A) is mixed with excess matrix (M) and dried • on a MALDI plate. • 2. Laser pulse ionizes matrix molecules. • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. AH+ Variable Ground Grid Grid +20 kV MALDI WRONG

  27. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE? • HOW TO CHOOSE A MATRIX? • HOW DOES IT WORK? • The SIMPLE explanation • The COMPLETE explanation

  28. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE? • HOW TO CHOOSE A MATRIX? • HOW DOES IT WORK? • The SIMPLE explanation • The COMPLETE explanation VERY COMPLEX: INVOLVES BOTH SOLID PHASE (PRIMARY IONS) AND GAS PHASE (SECONDARY IONS) PROCESSES AND ENERGETICS See work of Zenobi and Knochenmuss Mass Spectrometry Reviews, 1998, 17, 337–366 Mass Spectr., 2003, 17, 2034

  29. MALDI • Matrix Assisted Laser Desorption Ionization • WHAT MAKES A GOOD MATRIX? • HOW DO THEY LOOK LIKE? • HOW TO CHOOSE A MATRIX? • HOW DOES IT WORK? • The SIMPLE explanation • The COMPLETE explanation NOBODY REALLY KNOWS VERY COMPLEX: INVOLVES BOTH SOLID PHASE (PRIMARY IONS) AND GAS PHASE (SECONDARY IONS) PROCESSES AND ENERGETICS See work of Zenobi and Knochenmuss Mass Spectrometry Reviews, 1998, 17, 337–366 Mass Spectr., 2003, 17, 2034

  30. MALDI • WHAT DO MASS SPECTROMETRISTS DO WITH MALDI? (and/or electrosprays)

  31. MALDI • WHAT DO MASS SPECTROMETRISTS DO WITH MALDI? (and/or electrosprays)

  32. MALDI • WHAT DO MASS SPECTROMETRISTS DO WITH MALDI? (and/or electrosprays) • Dissociation studies • Mostly to sequence large biomolecules VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

  33. MALDI • WHAT DO MASS SPECTROMETRISTS DO WITH MALDI? (and/or electrosprays) • Dissociation studies • Mostly to sequence large biomolecules • Ion drift studies From Mike Bowers Lab (UCSB)

  34. Layout   • Introduction • WHY? WHY? WHY? WHAT? WHAT? WHAT? • Common ions production methods • Electrospray • Maldi • Cooling methods • Why cooling? • Supersonic expansions • Collisional cooling • Droplet evaporative cooling • Superfluid Helium Nanodroplet cooling • COUPLING A MALDI SOURCE TO A SUPERSONIC EXPANSION • Basic spectroscopy techniques applied toions • Advantages and drawbacks of working with ions (cf François Piuzzi lecture on spectroscopy of neutral species) • Examples of applications • IRMPD using Free Electron Lasers • Photodissociation electronic spectroscopy of protonated ions

  35. COOLING IONS • BIOLOGICAL PROCESSES TAKE PLACE AT ROOM TEMPERATURE…

  36. COOLING IONS • BIOLOGICAL PROCESSES TAKE PLACE AT ROOM TEMPERATURE… • Q : WHY DO SPECTROSCOPISTS WANT TO COOL DOWN MOLECULES/IONS TO TEMPERATURES DOWN TO FEW KELVINS?????

  37. COOLING IONS • BIOLOGICAL PROCESSES TAKE PLACE AT ROOM TEMPERATURE… • Q : WHY DO SPECTROSCOPISTS WANT TO COOL DOWN MOLECULES/IONS TO TEMPERATURES DOWN TO FEW KELVINS????? • A : TO BE ABLE TO ANALYZE THE SPECTRA!!!!

  38. COOLING IONS • How does cooling simplify spectra: energy ev S1 ev S0

  39. S1 S0 COOLING IONS • How does cooling simplify spectra: energy

  40. COOLING IONS • How does cooling simplify spectra: From Rizzo et al J. Am. Chem. Soc., 128 (9), 2816 -2817, 2006.

  41. COOLING IONS • How does cooling simplify spectra: ! From Rizzo et al J. Am. Chem. Soc., 128 (9), 2816 -2817, 2006.

  42. COOLING IONS • Besides simplifying the spectra, what are the effects of cooling large biomolecules? Biomolecules are large, floppy molecules that can adopt several conformations:

  43. COOLING IONS S1 UV energy The spectra of all the conformers are superposed S0

  44. COOLING IONS Potential energy Torsional angle Example : Phenylalanine

  45. COOLING IONS kT Potential energy Torsional angle

  46. kT COOLING IONS Potential energy Torsional angle

  47. kT COOLING IONS Potential energy Torsional angle

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