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

Experimental methods for the spectroscopic study of ionic

biomolecules in the gas phase

Pierre ÇarçabalLaboratoire de PhotoPhysique Moléculaire

CNRS – Orsay – France [email protected]

College of Photonics and Applications7-11 August 2006

Hanoi

mailto:[email protected]

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 to ions– 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

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 AFFECTMOLECULE’S ROLE?

- WHAT CAN GO WRONG?MOTOR - LIVING SYSTEM Gears - Isolated molecule

-WHAT ARE THE MINIMUMREQUIREMENTS TO DO THE JOB?

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)

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


OH

OH

N

H

C

O

• 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)

MOLECULAR ASSEMBLY

OH


OH

OH

N

H

C

O

• Hydrogen bonding plays a central role in ALL biological processes– What is the H bonding?

OH

MOLECULAR ASSEMBLY- single molecule → INTRAmolecular H bond- several molecules → INTERmolecular H bond

MOLECULAR ASSEMBLY


• 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

“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

http://www.whfreeman.com/biochem5/


• INFRARED SPECTROSCOPY IS A VERY SENSITIVE PROBE OF– MOLECULAR STRUCTURE

– HYDROGEN BONDING

molecule

O HOH

IR energy

molecule

O HO H

x

IR energy

OH stretching

NH stretchingCOH bending

3520 3540 3560 3580 3600 3620 3640 3660 3680

3520 3540 3560 3580 3600 3620 3640 3660 3680

wavenumber (cm -1)

3520 3540 3560 3580 3600 3620 3640 3660 3680

A

B

C

Example: Phenyl--D-mannopyranoside (pMan)

DOUBLE RESONANCE IR-UV

cG-g+0

ccG-g+3.2

cTt3.7

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


– WHY? WHY? WHY? WHAT? WHAT? WHAT?





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 (!!!!!)

Ions production methods

• TWO MAIN METHODS:– ELECTRO SPRAY IONISATION

– MALDI

Electro Spray Ionization

Analyte+

solvent

--

-

N2

analyser

3 – 6 kV

+

+

++

+

++ +

++

+

Mulitchargeddroplet

++

++

+++ +

+ ++

Solventevaporation

+

+++

++

+++

+

Coulombicexplosion

++

+

+++

Isolatedions

+

+

++

+

+

++ +

++

+

+-

-

--

-

-

---

----

++

+

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

MALDI

• Matrix Assisted Laser Desorption Ionization

Analyser

Pusher

Matrix ions

Analyte ions

LaserN2 (337 nm)Nd:Yag (355 nm)Nd:Yag (266nm)ExcimerCO2

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

MALDI

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

MALDISOME COMMON MALDIMATRICES:

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

Matrix Application

2,5-Dihydroxybenzoic acid (DHB) Peptides, proteins, lipids, and oligosaccarides

3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid)

Peptides, proteins, and glycoproteins

a-Cyano-4-hydroxycinnamic acid (CHCA)

Peptides, proteins, lipids, and oligonucleotides

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

MALDI

h

Laser

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+

+20 kV

Variable Ground Grid Grid

Sample plate

MALDI

h

Laser

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+

+20 kV

Variable Ground Grid Grid

Sample plate

WRONG

MALDI


» The SIMPLE explanation» The COMPLETE explanation

MALDI



VERY COMPLEX: INVOLVES BOTH SOLID PHASE(PRIMARY IONS) AND GAS PHASE (SECONDARY IONS)PROCESSES AND ENERGETICSSee work of Zenobi and Knochenmuss

Mass Spectrometry Reviews, 1998, 17, 337–366Mass Spectr., 2003, 17, 2034

MALDI



VERY COMPLEX: INVOLVES BOTH SOLID PHASE(PRIMARY IONS) AND GAS PHASE (SECONDARY IONS)PROCESSES AND ENERGETICSSee work of Zenobi and Knochenmuss

Mass Spectrometry Reviews, 1998, 17, 337–366Mass Spectr., 2003, 17, 2034

NOBODY REALLY

KNOWS

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

MALDI

• WHAT DO MASS SPECTROMETRISTS DO WITH MALDI? (and/or electrosprays)– Dissociation studies

» Mostly to sequence large biomolecules

MALDI

VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

http://www.logitheque.com/fiche.asp?I=11744





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)

COOLING IONS

• BIOLOGICAL PROCESSES TAKE PLACE AT ROOM TEMPERATURE…

COOLING IONS


• Q : WHY DO SPECTROSCOPISTS WANT TO COOL DOWN MOLECULES/IONS TO TEMPERATURES DOWN TO FEW KELVINS?????

COOLING IONS


• Q : WHY DO SPECTROSCOPISTS WANT TO COOL DOWN MOLECULES/IONS TO TEMPERATURES DOWN TO FEW KELVINS?????

• A : TO BE ABLE TO ANALYZE THE SPECTRA!!!!

COOLING IONS

• How does cooling simplify spectra:

S0

S1 ene

rgy

ev

ev

COOLING IONS


S0

S1 ene

rgy

COOLING IONS


From Rizzo et al J. Am. Chem. Soc., 128 (9), 2816 -2817, 2006.

COOLING IONS



!

COOLING IONS

• Besides simplifying the spectra, what are the effects of cooling large biomolecules?

Biomolecules are large, floppy moleculesthat can adopt several conformations:

COOLING IONS

S0

S1

UV energy

The spectra of all the conformersare superposed

COOLING IONSP

ote

ntia

l en

erg

y

Torsional angle

Example : Phenylalanine

COOLING IONSP

ote

ntia

l en

erg

y

Torsional angle

kT

• When one studies cold molecules, it may not be the same systems that at room temperature.

• The properties of cold systems are dictated by the POTENTIAL ENERGY while it is the FREE ENERGY that matters for high temperature systems (entropic effects).

• Depending on the cooling rate, we can end up with different conformational distributions

COOLING IONS

COOLING IONS

• SUPERSONIC EXPANSIONS

Carriergas

He/Ar/N2….

Few bars

VACCUUM CHAMBER10-4 mbar

COOLING IONS

• SUPERSONIC EXPANSIONS

COOLING IONS

• SUPERSONIC EXPANSIONS– INTERNAL ENERGY IS CONVERTED INTO

KINETIC ENERGY

N

vz

N

vz0

COOLING IONS

• SUPERSONIC EXPANSIONS– INTERNAL ENERGY IS CONVERTED INTO

KINETIC ENERGY» NEARLY MONOKINETIC BEAM (reduced Doppler effect)

» COOLING OF INTERNAL DEGREES OF FREEDOM

» CARRIER GAS ACTS AS A COOLING BATH FOR IMPURITIES (MOLECULES OF INTEREST)

– ZONE OF SILENCE» No interference with background gas

» Molecules studied in a collision free regime

– HIGH COLLISION RATE » Formation and stabilization of weakly bound complexes

COOLING IONS

• EFFECT OF SUPERSONIC COOLING ON SPECTRA

36300 36400 36500 36600 36700 36800 36900

wavenumber (cm-1)

i

ii

iii

36300 36400 36500 36600 36700 36800 36900

wavenumber (cm-1)

36300 36400 36500 36600 36700 36800 36900

wavenumber (cm-1)

i

ii

iii

-phenol fucose

COOLING IONS• COLLISIONAL COOLING

– Inelastic collisions between HOT MOLECULES and COLD ATOMS

– EFFICIENT FOR IONS WHICH CAN BE STORED FOR A LONG TIME IN ION TRAPS(quadrupole traps, ICR traps, 22-pole traps)

Cold (4-10 K) Heatoms

COOLING IONS• COLLISIONAL COOLING


COOLING IONS

• MORE EXOTIC COOLING METHODS

MICRODROPLET EVAPORATION

COOLING IONS

• MORE EXOTIC COOLING METHODS

SUPERFLUID HELIUM NANODROPLETS

T0 < 30KP0 ~ 50 barD = 5-20 μm

P ~ 10-5-10-4 Torr P ~ 10-7 Torr

Helium clusters production and dopping

~ 104 atoms0.38K

T0 < 30KP0 ~ 50 barD = 5-20 μm

P ~ 10-5-10-4 Torr P ~ 10-7 Torr

GAZ 1

0.2mTorr


T0 < 30KP0 ~ 50 barD = 5-20 μm

P ~ 10-5-10-4 Torr P ~ 10-7 Torr

GAZ 1

0.2mTorr

Quick thermalisation oftrapped species at 0.38K


T0 < 30KP0 ~ 50 barD = 5-20 μm

P ~ 10-5-10-4 Torr

~ 104 atoms0.38K

P ~ 10-7 Torr

GAZ 1

0.2mTorr

Oven

CaracterizationFIG

Mass specBolometer

GAZ 2


COOLING IONS

• COUPLING MALDI TO A SUPERSONIC EXPANSION

+ =?


COOLING IONS

High PressureDETECTION

10-4 mbar 10-6 mbar


COOLING IONS

Desorption laser

Detection

SPECTROSCOPY OF IONS

• IN PRINCIPLE, ONE CAN DO WITH IONS THE SAME SPECTROSCOPY AS FOR NEUTRALS– IR SPECTROSCOPY– UV/VIS SPECTROSCOPY– DOUBLE RESONANCE SPECTROSCOPY– ……

cf François Piuzzi’s lecture on the spectroscopy of neutral molecules

SPECTROSCOPY OF IONS

• BIG ADVANTAGES:– Using electromagnetic fields, you can do everything

you want with ions:» Accelerate them» Decelerate them» Deflect them» Trap them

– THEY ARE VERY EASY TO DETECT

• THE BIG PROBLEM:– To detect an optical transition, you have to fragment

them!» Require high power lasers

IRMPD

• InfraRed Multi Photon Dissociation

– Basic setup: IR tunablelaser

IRMPD

• InfraRed Multi Photon Dissociation– principle

IRMPD• Which laser can be used:

– Difference frequency IR generation» Table top» Easily tunable around 3 microns» Fairly high resolution (0.1 cm-1)» Relatively low photon fluence

– OPO» Table top» Easily tunable over a very wide range (2-10 microns)» Can be more powerful, but still…

– FEL» Cannot give photon in near IR (3 microns)» User facility» Give VERY high fluences between 5-20 microns

• With DFG and OPO, mostly limited to non-covalent (weakly bound) systems

IRMPD

Rizzo et al. J. Am. Chem. Soc., 128 (3), 905 -916, 2006

IRMPD

• WITH FEL LASERS : bare molecules can de dissociated!

Protonated Leucine ester

Ph. Maitre et al.International Journal of Mass Spectrometry Volumes 249-250 , 1 March 2006, Pages 14-20

http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6176&_auth=y&_acct=C000044719&_version=1&_urlVersion=0&_userid=922451&md5=06d163ecb90b10df0627f1b961bdc88d

http://www.sciencedirect.com/science?_ob=JournalURL&_cdi=6176&_auth=y&_acct=C000044719&_version=1&_urlVersion=0&_userid=922451&md5=06d163ecb90b10df0627f1b961bdc88d

http://www.sciencedirect.com/science?_ob=IssueURL&_tockey=%23TOC%236176%232006%23997509999%23617412%23FLA%23&_auth=y&view=c&_acct=C000044719&_version=1&_urlVersion=0&_userid=922451&md5=99a39f6479f28b93bd6d2c6ca53a9e37

SPECTROSCOPY OF COLD PROTONATED AMINO ACIDS

• Photodissociation of protonated peptidesphotone-

Selective dissociation

H transfer

( )REMEMBER

This model suggests a close relationship betweenfragmentation and structure


• Photodissociation of protonated peptides

• PERFORM SPECTROSCOPY ON COLD PROTONATED IONS AND CLUSTERS TO ESTABLISH THE RELATIONSHIP FRAGMENT CONFORMATION

TOF Einzel

Lens

Detector

10-4 mbar

VXY

10-6 mbarMALDILASER

MALDI+JET

Desorption



40 60 80 100 120 140 160 180 200 220 240 260

0

80

160pheH+fragments

91 a

mu

120

amu

mat

rix (

124

amu)

ion

sig

na

l (m

V)

m/z (amu)

pheH

+ (

166

amu)

TOF Einzel

Lens

VXY

MALDILASER

Reflectron

TOF

EinzelLens

VXY

Detector

MALDI+JET

Desorption

Argon supersonicexpansion

60 80 100 120 140 160 180

0

100

200

300

400

500

600io

n s

ign

al (

mV

)

m/z(amu)

ph

eH

+

40 60 80 100 120 140 160 180 200 220 240 260

0

80

160pheH+fragments

91 a

mu

120

amu

mat

rix (

124

amu)

ion

sig

nal

(m

V)

m/z (amu)

pheH

+ (

166

amu)

MASS FILTERING

TOF Einzel

Lens

VXY

MALDILASER

Reflectron

TOF

EinzelLens

VXY

Detector

MALDI+JET

Desorption


DISSOCIATIONLASER (UV)

TOF Einzel

Lens

VXY

MALDILASER

Reflectron

TOF

EinzelLens

VXY

Detector

MALDI+JET

Desorption


DISSOCIATIONLASER

IR spectroscopylaser

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

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