Fundamentals of Soft Ionization andMS Instrumentation

Ana Varela Coelhovarela@itqb.unl.pt

Mass Spectrometry LabAnalytical Services Unit

Index

• Mass spectrometers and itscomponents• Ionization methods: MALDI and ESI• Mass analysers: ToF, ToF/ToF and

Ion Trap

• Tadem mass spectrometry experiments

The mass spectrometer

The mass spectrum

The mass spectrum

So....A Mass Spectrometer

• generates gas-phase ions from asample

• separates them according to theirmass-to-charge ratio (m/z)

• produces a record of theirabundances

Mass-to-charge ratio(m/z)

• Unitless value used to refer to signals ina mass spectrum

• In principle, m/z unit is Thomson [Da/e]

Mass Spectrometer ComponentsSample Inlets

• Sample inlets• Direct infusion(DI) or

plate• Previous separation

method• Cromatography

(HPLC, GC)• Capillary

electrophoresis (CE)

MALDI plate

Mass Spectrometer ComponentsIon Sources

In general called ionization methods, but in fact are ionization (strong-inducefragmentation), desorption or desolvation (soft) methods

Gas-phase ionizationElectron ionization or impact(EI)Chemical ionization (CI)Desorption chemical ionization(DCI)Negative-ion chemicalionization (NCI)

Field desorption andionization

Field desorption (FD)Field ionization (FI)

Particle bombardmentFast atom bombardment (FAB)Secundary ion massspectrometry (SIMS)

Laser desorptionMatrix assistedlaser desorption(MALDI)

Atmospheric pressure ionization (API)

Electrospray ionization (ESI)Atmospheric pressure chemical

ionization (APCI)

Mass Spectrometer ComponentsIon Sources

In general called ionization methods, but in fact are ionization (strong-inducefragmentation), desorption or desolvation (soft) methods

Gas-phase ionizationElectron ionization or impact (EI)Chemical ionization (CI)Desorption chemical ionization(DCI)Negative-ion chemical ionization(NCI)

Field desorption andionization

Field desorption (FD)Field ionization (FI)

Particle bombardmentFast atom bombardment (FAB)Secundary ion mass spectrometry(SIMS)

Laser desorptionMatrix assisted laserdesorption (MALDI)

Atmospheric pressureionization (API)

Electrospray ionization (ESI)

Atmospheric pressure chemical ionization (APCI)

Mass Spectrometer ComponentsIon Sources

Reserpine spectraStrong ionization methods Soft ionization methods

MS

MS/MS

Mass Spectrometer ComponentsMass analyzers/designs

• Mass analysers (mass separation accordingm/z)• Ion Trap (3D and linear)• Time Of Fligth (TOF)• Orbitrap/FTICR

• Type of design defines type of instrument andsystem capabilities• Plate-MALDI-TOF• HPLC-ESI-quadrupole/ion trap• HPLC-plate-MALDI-TOF-TOF• HPLC-ESI-quadrupole-TOF• HPLC-ESI-orbitrap/FTICR

Mass Spectrometer ComponentsMass analyzers/designs

• Mass analysers (mass separation according m/z)• Ion Trap (3D and linear)• Time Of Fligth (TOF)• Orbitrap/FTICR

• Type of design defines type of instrument and systemcapabilities• Plate-MALDI-TOF• HPLC-ESI-quadrupole/ ion trap• HPLC-plate-MALDI-TOF-TOF• HPLC-ESI-quadrupole-TOF• HPLC-ESI-orbitrap/FTICR

Ion Sources (1)

Interaction between neutral species andgravity is weak, neutral molecular beam of mass range 100 to 200Daltons is gravity broadened by only 0.8 mm over a 1 km flight path

To achieve significant mass separation it isnecessary to ionize the species underinvestigation

The choice of the ionization method dependson the type of sample and informationrequired.

Soft Ion Sources-MALDI (1)

• Concept from Karas & Hillenkamp,1987(small organic compounds matrix)

• Tanaka,1988 (fine metal powder matrix)

Soft Ion Sources-MALDI (2)

• Analyte cocrystallized in matrix (UV-absorbing compound) irradiated bylaser, leading to sublimation andionization of analyte

• Ionizable molecules (1-2 charges),M>500Da (matriz ions visibles at lowM), low sensitivity to somecontaminants

Common MALDI Matrices (3)

Matrix-sample cocrystallization(4)

1 mm

1 mm

α-Cyano-4-hydroxycinnamic acid

2,5-Dihydroxybenzoic acid

2,5-Dihydroxybenzoic acidbad prep.

Courtesy of Dr Johan Gobom, Department of Clinical Neuroscience, University of Göteborg, Mølndal Hospital, Mølndal, Sweden

Soft Ion Sources-MALDI(5)

Soft Ion Sources-ESI (1)

• Design 1st described by Fenn, JB et al (1985)Nobel lecture 2002 "Electrospray Wings for Molecular Elephants"

• Transfer of analyte ionized species incondensed phase to gas phase as isolatedentities (3 step process: droplet formation, droplet shrinkage, gasous ionformation)

• Possible for ionizable molecules (activating collisions

possible), M range depends on charge state(usually multicharge), strong non-covalentinteractions are preserved

Soft Ion Sources-ESI (2)

• ESI interface • Droplet formation ESI interface

Soft ion sources-ESI vs MALDIRnase B ESI spectrum

Rnase B MALDI spectrum

Mass Analyzers

The choice of mass analyzer should be based on:applications

cost

performance desired

Mass analyzersTime-of-Fligth/TOF

• Ions generated by a pulsed laser, leavethe target at the same time, areaccelerated to Ec, travel along fligthtube

• Smaller m/z ions have lower tof (t),reach detector before larger ions -MSspectrum

Mass analyzers TOF

Mass analyzersReflectron TOF

• A reflectron ion mirror reverses the flightpath of the ions, corrects for differencesin the Ec of ions with the same m/z

• Better resolution, but lower sensitivity

• For m/z < 5 000 Da

Mass analyzersReflectron TOF

Linear vs reflectron

MALDI-TOF-TOF

Tandem MS or MS/MS

TOF-TOF Mass Analyzers MS/MS

• Isolation molecular ion (1st TOF)• Fragmentation (collision cell)• Scan fragment ions (2nd TOF)

Aim: selective structural study of an ionStep 1: Isolation of molecular ionStep 2: Fragmentation forms fragment ions

MALDI-TOF-TOF

Mass analyzers 3D Ion Trap• Ions trapped in 3D electric field• Ions are captured until limite

trap charge space• A bath gas (He) helps to

contain ions in the trap• Ions subject to additional

electric field which ejects themsequentially

• Ions reach the detector-MSspectrum

ESI-ION TRAP

Mass analyzersLinear Ion Trap

• High sensitivity

• Low duty cycletime

MS applications• M determination and structural characterization of inorganic,

organic and biological compounds• Characterization and quality control of recombinant proteins• Detection/characterization posttranslational modifications• Quantitative MS (using stable isotopes)• Characterization of non-covalent complexes• Folding-unfolding, conformation exchange studies• Microorganism identification• Metabolism studies• Protein identification