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Fundamentals of Soft Ionization andMS Instrumentation
Ana Varela [email protected]
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
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
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-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
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 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
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
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
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