Fundamentals of Soft Ionization and MS Instrumentation
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1 Fundamentals of Soft Ionization and MS Instrumentation Ana Varela Coelho Mass Spectrometry Lab Analytical Services Unit
2 Index Mass spectrometers and its components Ionization methods: MALDI and ESI Mass analysers: ToF, ToF/ToF and Ion Trap Tadem mass spectrometry experiments
3 The mass spectrometer
4 The mass spectrum
5 The mass spectrum
6 So...A Mass Spectrometer generates gas-phase ions from a sample separates them according to their mass-to-charge ratio (m/z) produces a record of their abundances
7 Mass-to-charge ratio (m/z) Unitless value used to refer to signals in a mass spectrum In principle, m/z unit is Thomson [Da/e]
8 Mass Spectrometer Components Sample Inlets Sample inlets Direct infusion(di) or plate Previous separation method Cromatography (HPLC, GC) Capillary electrophoresis (CE) MALDI plate
9 Mass Spectrometer Components Ion Sources In general called ionization methods, but in fact are ionization (strong-induce fragmentation), desorption or desolvation (soft) methods Gas-phase ionization Electron ionization or impact (EI) Chemical ionization (CI) Desorption chemical ionization (DCI) Negative-ion chemical ionization (NCI) Field desorption and ionization Field desorption (FD) Field ionization (FI) Particle bombardment Fast atom bombardment (FAB) Secundary ion mass spectrometry (SIMS) Atmospheric pressure ionization (API) Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Laser desorption Matrix assisted laser desorption (MALDI)
10 Mass Spectrometer Components Ion Sources In general called ionization methods, but in fact are ionization (strong-induce fragmentation), desorption or desolvation (soft) methods Gas-phase ionization Electron ionization or impact (EI) Chemical ionization (CI) Desorption chemical ionization (DCI) Negative-ion chemical ionization (NCI) Field desorption and ionization Field desorption (FD) Field ionization (FI) Particle bombardment Fast atom bombardment (FAB) Secundary ion mass spectrometry (SIMS) Atmospheric pressure ionization (API) Electrospray ionization (ESI) Atmospheric pressure chemical ionization (APCI) Laser desorption Matrix assisted laser desorption (MALDI)
11 Mass Spectrometer Components Ion Sources Reserpine spectra Strong ionization methods MS Soft ionization methods MS/MS
12 Mass Spectrometer Components Mass 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 system capabilities Plate-MALDI-TOF HPLC-ESI-quadrupole/ion trap HPLC-plate-MALDI-TOF-TOF HPLC-ESI-quadrupole-TOF HPLC-ESI-orbitrap/FTICR
13 Mass Spectrometer Components Mass 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 system capabilities Plate-MALDI-TOF HPLC-ESI-quadrupole/ ion trap HPLC-plate-MALDI-TOF-TOF HPLC-ESI-quadrupole-TOF HPLC-ESI-orbitrap/FTICR
14 Ion Sources (1) Interaction between neutral species and gravity is weak, neutral molecular beam of mass range 100 to 200 Daltons is gravity broadened by only 0.8 mm over a 1 km flight path To achieve significant mass separation it is necessary to ionize the species under investigation The choice of the ionization method depends on the type of sample and information required.
15 Soft Ion Sources-MALDI (1) Concept from Karas & Hillenkamp,1987 (small organic compounds matrix) Tanaka,1988 (fine metal powder matrix)
16 Soft Ion Sources-MALDI (2) Analyte cocrystallized in matrix (UVabsorbing compound) irradiated by laser, leading to sublimation and ionization of analyte Ionizable molecules (1-2 charges), M>500Da (matriz ions visibles at low M), low sensitivity to some contaminants
17 Common MALDI Matrices (3)
18 Matrix-sample cocrystallization(4) 1 mm 2,5-Dihydroxybenzoic acid 1 mm α-cyano-4-hydroxycinnamic acid 2,5-Dihydroxybenzoic acid bad prep. Courtesy of Dr Johan Gobom, Department of Clinical Neuroscience, University of Göteborg, Mølndal Hospital, Mølndal, Sweden
19 Soft Ion Sources-MALDI(5)
20 Soft Ion Sources-ESI (1) Design 1 st described by Fenn, JB et al (1985) Nobel lecture 2002 "Electrospray Wings for Molecular Elephants" Transfer of analyte ionized species in condensed phase to gas phase as isolated entities (3 step process: droplet formation, droplet shrinkage, gasous ion formation) Possible for ionizable molecules (activating collisions possible), M range depends on charge state (usually multicharge), strong non-covalent interactions are preserved
21 Soft Ion Sources-ESI (2) ESI interface Droplet formation ESI interface
22 Soft ion sources-esi vs MALDI Rnase B ESI spectrum Rnase B MALDI spectrum
23 Mass Analyzers The choice of mass analyzer should be based on: applications cost performance desired
24 Mass analyzers Time-of-Fligth/TOF Ions generated by a pulsed laser, leave the target at the same time, are accelerated to Ec, travel along fligth tube Smaller m/z ions have lower tof (t), reach detector before larger ions -MS spectrum
25 Mass analyzers TOF
26 Mass analyzers Reflectron TOF A reflectron ion mirror reverses the flight path of the ions, corrects for differences in the Ec of ions with the same m/z Better resolution, but lower sensitivity For m/z < Da
27 Mass analyzers Reflectron TOF
28 Linear vs reflectron
29 MALDI-TOF-TOF
30 Tandem MS or MS/MS Aim: selective structural study of an ion Step 1: Isolation of molecular ion Step 2: Fragmentation forms fragment ions TOF-TOF Mass Analyzers MS/MS Isolation molecular ion (1st TOF) Fragmentation (collision cell) Scan fragment ions (2nd TOF)
31 MALDI-TOF-TOF
32 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 them sequentially Ions reach the detector-ms spectrum
33 ESI-ION TRAP
34 Mass analyzers Linear Ion Trap High sensitivity Low duty cycle time
35 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
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