Fundamentals of Mass spectrometry

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Fundamentals of Mass spectrometry
February 6, 2008 Case Center for Proteomics
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What is mass spectrometry?

• Mass spectrometry is an instrumental approach
  that allows for the mass measurement of ions
  derived from molecules.
• Mass spectrometers are capable of forming,
  separating and detecting molecular ions on their
  mass-to-charge ratio.

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Sample Introduction
Sample
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Vacuum in MS
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Sample Introduction - Ionization tasks

• Need to transfer sample from atmospheric pressure
  to vacuum
• Samples solid, liquid, gas
• Need to create ions in the gas phase
• Gas ionize
• Liquid evaporate, than ionize OR
• both at the same time (spray)
• Solid sublime, than ionize OR
• both at the same time (desorption)

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• Gaseous samples
• Easy to transfer from A.P. to vacuum
• Liquid/solid (high volatility) vapor can be used
  as well must be free of air (freeze-pump-thaw
  cycles)
• Controlled flow leak valves, capillaries
• Mixed with carrier gas
• GC interface (GC/MS)

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• Liquid and solid samples
• Liquid samples analyte solutions spray
  ionization techniques
• Solid samples Solids probe
• Low volatility can be put in the vacuum
• Vacuum lock to keep high vacuum intact
• Evaporation by heating, then ionization OR
• Desorption by laser / particle beam

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Once analyte molecules are in the gas phase they
can easily be ionized

• Electron Impact (EI) and Chemical Ionization (CI)
• FAB-fast atom bombardment ionization
• MALDI-TOF
• ESI
• Electron-Capture ionization

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(No Transcript)
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EI(top) vs CI of ephedrine
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EI and CI Summary

• EI gives extensive fragmentation (70eV) but has
  extensive database for fingerprinting
• CI is more suitable for molecular weight
  determination but structural info is limited
• Alternate EI/CI can combine advantages
• Excellent sensitivity and natural GC/MS
  interface
• Major limitation MW lt 1000 Da, volatility must
  be high, not very suitable for most biological
  compounds
• See Interpretation of Mass Spectra by
  McLafferty and Turecek (FRL) for EI/CI of organic
  molecules

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Fast Atom Bombardment (FAB)
To mass analyzer
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FAB

• Higher mass range (7 kDa)
• Easy to get molecular ion MH or (MNa), little
  fragmentation
• Can be done on Hi-Res mass spectrometers
• Both positive and negative ion modes possible

• Bad sensitivity, especially at high masses
• High background of matrix peaks
• Sample must be soluble in matrix
• Not very useful for non-polar species

• Was a big step up from EI/CI for the analysis
  of
• biomolecules, but now is almost obsolete

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Matrix-Assisted Laser Desorption/Ionization
(MALDI)
MH
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MALDI

• Tanaki, Chemistry Nobel Prize 2002 (shared)
• Karas Hillenkamp, 1985-1988
• Laser UV _at_ 337, 355 or 266 nm
• or IR _at_ 3 or 10 mm
• Matrix must absorb well in UV (IR) region
• Subst. aromatic acids, alcohols
• Sample is mixed with matrix solution and dried,
  almost anything can be analyzed, including very
  high MW compounds

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Typical MALDI matricies

• UV MALDI
• Sinapinic acid (SA) 3,5-Dimethoxy-4-hydroxycinnam
  ic acid
• DHB 2,5-Dihydroxybenzoic acid
• CHCA a-Cyano-4-hydroxycinnamic acid
• Ferulic acid (FA) 4-Hydroxy-
• 3-methoxycinnamic acid
• p-Nitrophenol

• Solvent
• Water/ ACN in diffferent proportions (0-100)
• Sometimes Ethanol or other organic solvents

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Merits of MALDI

• Low resolution
• Matrix background for MW lt 1000 Da
• (DIOS no matrix
• and 700 amol sensitivity !)
• Other surfaces are being tested

• High accessible mass range (up to 1,000,000)
• Femtomole and better sensitivity
• Soft ionization almost no fragmentation
• Very good for mixture analysis, can work in the
  presence of salts

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Electrospray Ionization (ESI)
lt- 5 kV dif. -gt
Analyte solution
Analyte solution, 5 mL/min
-gtIons to MS
Fenn and others, 1984 Fenn, Nobel Prize 2002
(shared)
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Ion Desolvation in ESI
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Comparison of Ionization Methods
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Mass Analysis
Sorting of Ions
Ion Detection

Ion Source
Mass Analyzer
















Based on their m/z
Mass Spectrum
First mass analyzers early 1900s
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Performance of mass analyzer

• Mass range (low to high m/z)
• Resolution (separation of two adjacent peaks in
  the mass spectrum)
• Detection sensitivity (lowest amount of analyte
  that can be detected)
• Scan speed (time required to scan all ions in the
  m/z range of interest)

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Resolution
(Resolving Power) R M/DM Where
DMM2-M1 (2 peaks) or FWHM (1 peak)
M2
M1
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Types of mass analyzers

• Magnetic / Sector
• Quadrupole (Q)
• Quadrupole Ion Trap (IT), Linear Trap (LT)
• Time-of-Flight (TOF)
• Fourier-Transform Ion Cyclotron Resonance (FT-ICR
  or FT-MS)
• Ion Mobility

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Magnetic mass analyzer
Kinetic energy zV ½ mv2 In magnetic field
mv2/r zvB Combine m/z B2r2/2V
Alternatively, with a fixed slit position, Scan
magnetic field B gt mass spectrum
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Merits of sector instruments

• Benefits
• High resolution (gt100, 000)
• Good mass range (gt15,000 m/z)
• Reasonable scan speed and dynamic range, good
  mass accuracy
• High sensitivity
• CID MS/MS spectra are very reproducible
• Limitations
• Huge cost, hard to operate
• Low ion transmission efficiency
• Applications
• All organic MS analysis methods
• Accurate mass measurements
• Quantitation
• Isotope ratio measurements

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Quadrupole mass filter
Combination of DC and RF voltages on 4
rods Allows to pass ions of only certain m/z
Scan RF field to obtain MS
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RF-only Quadrupole

• In the absence of DC field, quadrupole operates
  as a wide-band mass filter. It can be used as an
  ion guide or reaction chamber
• Higher-order multipoles (hexapoles, octopoles,
  etc.) are used for ion-guiding and
  ion-accumulating purposes

DC-only Quadrupole

• In the absence of RF field, quadrupole operates
  as a lens element in some ion optics designs.

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Quadrupole features

• Benefits
• Upper mass limit up to 4000 m/z
• m 0.136 V / (f2r2)
• Resolution typically low (unit mass) R ? mf2L2/
  (zV)
• Can tolerate pressure up to 5x10-5 Torr
• Good for GC, LC, CE interfaces
• Natural analyzer for ESI sources
• Low cost gt most popular mass analyzer

• Limitations
• Limited resolution
• Peak heights variable as a function of mass
• CID depends on energy, collision gas, pressure

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Quadrupole Ion Traps
Paul,Steinwedel 1953
Principle similar to Quadrupole MS
Ions are trapped in mTorr of He
-V pulses open V closed to inject ions
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IT Modes of operation
Ion storage Ring electrode RF only Endcaps
grounded Ions of all m/z are trapped
m/z 4V / (qmaxw2r02)
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Trapping Ions in the Axial (Z) Direction
3 130 VDC
Creates a Potential Energy Well to Confine the
Ions
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Heliums Role as a Damping Gas
Potential Well
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The Effects of No Helium

Helium flowing into trap
Helium shut off and not flowing into trap
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Linear Ion Trap Stability Diagram

• Ions stability depends upon
• m/z
• Frequency of fundamental RF
• Amplitude of the V on the ring electrode
• Ion trap size

1.0
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Mass Selective Instability
q - Axis
At a particular RF voltage, every mass moves
with a specific (secular) frequency
q .908

• Ramp RF, Ions Leave Low m/z to High m/z
• CID RF resonance excitation voltage to end caps
  electrodes to enable energetic collisions with He

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Ion Traps merits

• Easily coupled to EI/CI (GC) ESI (LC, CE)
  through ion guides other mass analyzers (TOF,
  Ion Mobility)
• Low cost, good sensitivity
• R5,000 (gt30,000 at low scan speeds)
• Mass range 2-4,000 m/z (gt20,000)
• Ion chemistry, MSn capability

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Fourier-transform Ion Cyclotron Resonance Mass
Spectrometer
Another type of ion-trapping MS
Ions are trapped by combination of electric(DC)
and magnetic fields
Ions can be excited by applying RF voltage to
transmitter plates Ion selection, MSn, Ion
detection
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Ion excitation
w B z/m
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Ion Detection in FT-MS
Unique type of ion detection, one measures not
the ion but image current. Non-destructive !
Receiver plate
Receiver plate
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Fourier transformation
Pulse covers whole frequency range

• B z/m or
• f 15357 B z/m
• kHz T

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Benefits High Resolution
R lt B T z/m
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FT-MS figures of merit

• Ultrahigh resolution (gt1,000,000 for
  biomolecules, 108 achieved for m/z18)
• Unsurpassed mass accuracy, good mass range
  (gt20,000 m/z with 4.7 T magnet)
• Extremely versatile in terms of ion chemistry
  (MSn, dissociation, ion-molecule reactions), ions
  can be trapped for days!
• Cost more than one would like
• Require low pressures (lt10-7 Torr)
• Supercon. magnets require space/LHe/LN2

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Time-of-Flight MS
mv2/2 zV v (2zV/m)1/2 vL/t t L/v
L(m/2zV)1/2
Naturally couples to MALDI, can be adopted to ESI
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Resolution in TOF
Rm/Dmt/Dt
Problems Temporal and spatial ion distribution
Use short pulse and high extraction V (gt20 kV) R
used to be lt500, now lt 5000
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Reflectron TOF
(reflectron)
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Delayed Extraction
In a regular TOF experiment, ions are extracted
and detected promptly before they can fragment
gt Lack of structural information Ion extraction
can be time-delayed (DE) to give the ions time
to fragment. (Post-source decay PSD).
Fragments. DE not only allows detection of
source-formed fragment ions but also improves
mass accuracy and resolution.
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Merits of TOF

• Virtually unlimited mass range
• Very high scan speed (as low as 25 ms)
• High ion transmission, low detection limits
• Simple design, low cost
• Decent resolution
• Pulsed mode, hard to
• interface with ESI

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TOF summary

• Unlimited mass range in linear mode
• Resolution 3000 (L) 10,000 (R), more
• Extremely quick scan
• Excellent sensitivity
• Simple design, portability
• DE for structure elucidation
• OA for ESI interface

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Mass Analyzers Comparison