Chemical Analysis by Mass Spectrometry

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Chemical Analysis by Mass Spectrometry

• Dr Phil Mortimer
• Chemistry Department Mass Spectrometry Facility
• 410-516-5552
• mass.spec_at_jhu.edu

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Recommended Reading The Expanding Role of
mass Spectrometry in Biotechnology Gary
Siuzdak, MCC Press, San Diego, ISBN
0-9742451-0-0 Ionization Methods in Organic
Mass Spectrometry Alison Ashcroft, RSC,
Cambridge, UK, ISBN 0-85404-570-8 Practical
Organic Mass Spectrometry 2nd Edn J R Chapman,
Wiley, Chichester, UK, ISBN 0-471-95831-X Spectr
oscopic Methods in Organic Chemistry 4th Edn D H
Williams, I Fleming, McGraw-Hill, ISBN
0-07-707212-X
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Chemistry 101

• All chemical substances are combinations of
  atoms.
• Atoms of different elements have different
  masses (H 1, C 12, O 16, S 32, etc.)
• An element is a substance that cannot be broken
  down into a simpler species by chemical means -
  has a unique atomic number corresponding to the
  number of protons in the nucleus
• Different atoms combine in different ways to
  form molecular sub-units called functional
  groups.

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Chemistry 101

• Mass of each group is the combined mass of the
  atoms forming the group (often unique)
• e.g. phenyl (C6H5) mass 77, methyl (CH3) mass
  15, etc.
• So- If you break molecule up into constituent
  groups and measure the mass of the individual
  fragments (using MS) - Can determine what groups
  are present in the original molecule and how they
  are combined together
• ? Can work out molecular structure

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What is Mass Spectrometry? Mass spectrometry
is a powerful technique for chemical analysis
that is used to identify unknown compounds, to
quantify known compounds, and to elucidate
molecular structure Principle of operation A
Mass spectrometer is a Molecule
Smasher Measures molecular and atomic masses
of whole molecules, molecular fragments and atoms
by generation and detection of the corresponding
gas phase ions, separated according to their
mass-to-charge ratio (m/z). Measured masses
correspond to molecular structure and atomic
composition of parent molecule allows
determination and elucidation of molecular
structure.
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What is Mass Spectrometry? May also be used
for quantitation of molecular species. Very
sensitive technique - Works with minute
quantities of samples (as low as 10-12g, 10-15
moles) and is easily interfaced with
chromatographic separation methods for
identification of components in a mixture Mass
spectrometry provides valuable information to a
wide range of professionals chemists,
biologists, physicians, astronomers,
environmental health specialists, to name a few.
Limitation is a Destructive technique
cannot reclaim sample
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• What is Mass Spectrometry Used For?
• Chemical Analysis and Identification
• Some Typical Applications
• Enviromental Monitoring and Analysis (soil,
  water and air pollutants, water quality, etc.)
• Geochemistry age determination, Soil and rock
  Composition, Oil and Gas surveying
• Chemical and Petrochemical industry Quality
  control
• Applications in Biotechnology
• Identify structures of biomolecules, such as
  carbohydrates, nucleic acids
• Sequence biopolymers such as proteins and
  oligosaccharides
• Determination of drug metabolic pathways

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• How Does it Work?
• Generate spectrum by separating gas phase ions
  of different mass to charge ratio (m/z)
• mmolecular or atomic mass, z electrostatic
  charge unit
• In many cases (such as small molecules), z 1
• ? measured m/z mass of fragment
• But this is not always true
• For large bio-molecules analysed by electrospray
  (ESI), z gt1
• What happens in this case?

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Multiple Charging Consider a peptide with MW
of 10000 With ESI-MS, charges by H
addition M nH ? MnHn Resultant ions
formed are - When z 1 m/z (100001)/1
10001 When z 2 m/z (100002)/2
5002 When z 3 m/z (100003)/3
3334.3 When z 4 m/z (100004)/4
2501 When z 5 m/z (100005)/5 2001
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Figure from The Expanding Role of MS in
Bio-technology G . Siuzdak
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• Multiple Charging
• Advantage in that allows measurement of high mass
  ions with instruments of limited m/z range.
• Particularly true for ESI-MS Advantage for
  analysis of high mass samples that take multiple
  charges brings sample m/z down into measurable
  range of MS
• Computer Algorithms deconvolute m/z to original
  mass.

Figure from The Expanding Role of MS in
Biotechnology G . Siuzdak
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Mass Measurement Mass Spectrometers measure
isotopic mass. They DO NOT measure average
molecular mass!! (MW) e.g For a molecule with
empirical formula C60H122N20O16S2 Average MW
1443.8857 (weighted average for each
isotope) Exact mass 1442.8788 (exact mass
of most abundant isotope) Nominal mass
1442 (integer mass of most abundant
isotope) Illustrated on next Slide
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Resolution Influences achievable
precision and accuracy of measurement
Figure from The Expanding Role of MS in
Bio-technology G . Siuzdak
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Resolution Influences
achievable precision and accuracy of measurement
R ?M/M Often expressed in ppm R (?M/M) x106
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Isotope Patterns
Isotope patterns useful for identifying presence
of certain elements Particularly useful for
SMALL molecules
Figure from The Expanding Role of MS in
Bio-technology G . Siuzdak
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What is a Mass Spectrometer? Many different
types each has different advantages, draw-backs
and applications All consist of 4 major sections
linked together Inlet Ionization source
Analyser Detector All sections usually
maintained under high vacuum All functions of
instrument control, sample acquisition and data
processing under computer control Data system
and Computer Control is often overlooked most
significant advance in MS allows 24/7
automation and development of modern powerful
analytical techniques.
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What is a Mass Spectrometer?

• All Instruments Have
• Sample Inlet
• Ion Source
• Mass Analyzer
• Detector
• Data System

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Mass spectrometry
How does it work?
accelerate
separate
ionise
4000 V
0 V
e-
Magnetic and/or electric field
e-
heavy
vacuum
light
vapourise
e-
e-
sample
A
B
C
e-
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Analyser Types What is the analyser? Analyser
is the section of instrument that separates ions
of different m/z Many Different
technologies Magnetic Sector, Quadrupole, Ion
Trap, ToF All based on momentum separation
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Analyser Types Magnetic sector Easiest
Conceptually to understand Separate
electromagnetically Electromagnetic
Prism Usually combined with ESA (energy
focusing device) - enables high mass resolution
(Double Focusing Instrument) makes high
accuracy mass measurements possible Large
(Heavy!!), Expensive to operate Comparatively
slow scan rates High Skill level required to
operate and maintain Self-service use by users
not possible
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Analyser Types Quadrupole Smaller, cheaper
computer controlled Self service operation by
trained users possible Electrostatic momentum
separation by superimposed rf and dc
voltages Rapid scan rates enables measurement
of transient samples introduced from
chromatographic systems (GC, LC) Lower
resolution accurate mass NOT possible
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Analyser Types Quadrupole ion Trap Derivative
of Quadrupole cheap, small, rapid
scanning Again, electrostatic momentum
separation by rf and dc voltages Lower
resolution accurate mass not possible BUT
have ion trapping ability can store and
selectively eject ions Ions can be subjected to
fragmented by CID and daughter ions
analysed Allows MS-MS or MSn (Multiple levels of
storage and trapping) Can perform both molecular
ion analysis and structural determination
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Analyser Types Quadrupole ion Trap
3 Electrode system 2 x Endcap and 1x Ring
Electrode Now have recent develpoment of Linear
Ion Trap and orbitrap Developments on same theme.
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Analyser Types Quadrupole ion Trap
Bruker HCT
Ion Trap is very small most of instrument is
ion guides into the trap itself
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Analyser Types Time of Flight (ToF) Conceptual
diagram!!!
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Analyser Types Time of Flight (ToF) Velocity
separation - E mv2 Ion packet given constant
KE ions of heavier mass take longer to pass
down drift tube and reach detector Conceptually
easy Allows very large masses to be measured
(500,000Da) E 1/2mv2
Time flight of ions through drift tube Ions of
larger mass take longer to reach detector for
constant E
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Mass Spectrometer Instrument Design Different
types of Ionization source EI, CI, FAB, ESI,
Maldi, (APCI, DESI, DART) (Also sources for
inorganic analysis ICP, GD, etc.) Different
types of analyser Magnetic Sector, Quadrupole,
Ion Trap, ToF Different sources and analysers
have different properties, advantages and
disadvantages Selection of appropriate
ionization method and analyzer are critical and
defines MS applications. Wide range of MS
applications
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Development of Mass Spectrometry Until 1980s,
most mass spec geared primarily towards
traditional chemical analysis (small
molecules) - MS primarily conducted using EI
ionisation unchanged since 30s and 40s From
1980s, start to have shift in focus towards
analysis of samples that are larger and more
bio-molecular in character Such samples are
often more delicate and easily fragmented. This
results in the development of softer ionisation
techniques and analysers capable of extended mass
ranges. Allows MS determination of high mass
parent ions (such as intact proteins,
etc.). Strongly influences development of
Proteomics field
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Electron Impact (EI) Mass Spectrometry Up until
1980s, most mass spec is chemical analysis -
performed using EI ionisation Bombard gaseous
sample with high energy (70eV) e- Results in
ejection of e- from target molecule to form gas
phase ion species which is then passed to
analyser for analysis. e- M -gt 2e- M Sample
normally introduced via heated probe, GC, or leak
(frit) inlet
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• Electron Impact (EI) Mass Spectrometry
• Problems with EI ionisation
• requires sample be in the gas phase before
  ionisation
• - limits samples to those already existing in
  the gas phase or thermally stable samples that
  are easily volatised (for probe introduction)
• 2) High Energy (Hard) Ionisation lots of
  excess energy given to target causes
  fragmentation to lose energy and become stable
  resulting in lots of characteristic fragments
  ions, but little parent ion (useful for
  structural characterisation).

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Electron Impact (EI) Mass Spectrometry
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• Overcoming problems with EI-MS Use of CI
• How to overcome limitations?
• Derivatize sample to make more volatile and
  thermally stable derivative that can be analysed
  by EI
• 2) Develop other ionisation techniques using
  lower ionisation energies and other means of
  introducing sample.
• Intermediate method was Chemical Ionisation (CI)
• Uses bath gas (CH3/NH4/CH3(CH2)2CH3) to protonate
  sample
• Often forms MH
• Still only applicable to volatile or Thermally
  stable samples.

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CI-MS Comparison of EI and CI spectra
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FAB-Mass Spectrometry Subsequent development of
FAB (Fast Atom Bombardment) Still used for small
delicate molecules Dissolve sample in liquid
matrix and place on target Bombard with beam of
fast atoms or ions (Xe or Cs) Have secondary
ion emission Low energy protonation of target
molecules very little excess energy little
fragmentation readily observe parent ions. Now
were getting somewhere.
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FAB-Mass Spectrometry
Problems with FAB Slow, Labor intensive, Very
skilled. Matrix interference at low mass
Generally observe MH (ve ion mode) OR M-H
(-ve ion mode)
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Current Mass Spectrometry Biochemical
MS Today, majority of MS is of bio-chemiccal /
biological samples performed using either
Electrospray MS or Maldi-toF MS. Other methods
exist, but these perform bulk of the work Will
concentrate on these for the rest of the
lecture. Both are soft (low energy) ionisation
methods that usually yield little fragmentation
and so are useful for determination of parent
mass of delicate molecules. Both are condensed
phase techniques and require that samples are
soluble.
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Electrospray Mass Spectrometry (ESI-MS) Solution
phase technique - Can analyse both ve and ve
ions (but not simultaneously) Samples usually
dissolved in moderately polar solvent Typically
MeOH or MeCN, often mixed H2O (up to 80) DO
NOT USE DMF, DMSO, THF, etc Do NOT use
involatile buffers. Typical concentration 1-10uM
(can be 20nM-50uM depending on sample) Usually
requires addition of volatile buffer
(0.1-1) Typically AcOH or TFA (ve ion) /
NH4OH (-ve ion)
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Electrospray Mass Spectrometry (ESI-MS) How does
it work?
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Electrospray Mass Spectrometry (ESI-MS) How does
it work?
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Electrospray Mass Spectrometry (ESI-MS)
Thermo-Finnigan LCQ-Deca ESI-Ion Trap with LC
System
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Electrospray Mass Spectrometry (ESI-MS) Different
versions of ESI (On-Axis / Orthoganal / Off
Axis) Advantages Soft ionisation limited
fragmentation Multiple charging with peptides /
proteins / oligionucleotides (Analysis of
molecules with MW gt mass range of instrument) Can
be linked with LC acts as inlet allows MS
identification of components of
mixtures Automated high throughput analysis of
biological samples 24/7 Can be coupled with
many analysers IT/Quadrupole /ICR / Orbitrap
vast range of different types of analysis
possible
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Electrospray Mass Spectrometry (ESI-MS)
Can Deconvolute mass spectra as previously
discussed
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MALDI-ToF Mass Spectrometry Relatively simple
technique Soft ionisation method that can be
used to volatilise large macromolecules with
minimum fragmentation Gives less multiple
charging than ESI Samples co-deposited on target
plate with matrix (and often an additive) and
allowed to dry. Many samples can be on
plate. Plate inserted into instrument vacuum
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MALDI-ToF Mass Spectrometry Target irradiated by
UV laser. Causes vaporisation of matrix and
supersonic expansion of plume Dried sample is
launched into the gas phase as matrix is
vaporised UV energy absorbed by matrix causes it
to dissociate and typically transfers a proton to
sample molecule within the plume to form MH Now
have protonated target, which is accelerated into
analyser for seperation and detection
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MALDI-ToF Mass Spectrometry
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MALDI-ToF Mass Spectrometry
Most MALDI-ToF are reflectron instruments Reflect
ron is energy focusing device (ion
mirror) Increases resolution (and mass accuracy)
but limits mass range Linear ToF has low
resolution but high mass range (up to m/z
300,000) Many Instruments are now ToF/ToF Can
do MS/MS experiments
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MALDI-ToF Mass Spectrometry
Typical Current State of the Art
Maldi-ToF Bruker Autoflex Now available as
Tof/ToF Easy to use walk up use after
training. Highly automated Now can be used for
imaging of Tissue samples
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MALDI-ToF Mass Spectrometry - Conditions Suggest
ed concentrations 10 pmol _at_ lt10 000 Da
(pure) 100 pmol _at_ gt50 000 Da (pure) 10 1
Ratio of Matrix Sample (20nM-50uM of sample
typically 1-10uM) Several methods of target
prep Multiple layer / co-mixed Spot 0.5uL of
mixture on spot and allow to dry Analysis very
dependant upon sample preparation
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MALDI-ToF Mass Spectrometry - Matrices
Matrix Application
a-Cyano-4-hydroxycinnamic acid (CCA) peptides
3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) proteins
2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars
3-Hydroxypicolinic acid (HPA) oligonucleotides
Dithranol (anthralin) polymers
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• MALDI Contamination Limits
• Analysis is relatively insensitive to
  contaminants.
• Phosphate 20 mM EDTA 1 mM
• Detergents 0.1 Glycine 20 mM
• Glycerol 2 Sodium Citrate 20 mM
• Buffer (Tris)50 mM K phosphate 25 mM
• Guanidine 1 M Na phosphate 0.1M
• Na azide 1 Octyl glucoside 0.3
• SDS 0.05 Ammon. Bicarb. 0.1M
• Suggested concentrations
• 10 pmol _at_ lt10 000 Da (pure)
• 100 pmol _at_ gt50 000 Da (pure)

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• MALDI Characteristics
• Maldi-ToF Generally results in broader peak
  envelope than ESI
• This is particularly true at high mass.
• Low mass Maldi-ToF (lt20,000Da) can use
  reflectron get high resolution (Rgt10,000)
• High MW Maldi requires use of linear mode
  lower resolution Higher Mass range (up to
  500,000Da
• Maldi-ToF generally results in generation of
  singly charged species (z 1)
• However, often requires desalting, otherwise
  have broad mass envelop addition due to multiple
  slated peaks forming particularly prevalent for
  proteins

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• MALDI Characteristics
• Analysis is rapid therefore, is often used for
  high throughput analysis and screening
  applications many samples on one plate.
• Sensitivity enhanced by using AnchorChip
  Plates concentrates sample solution in small
  spot
• Low mass spectra (lt500MW) can be inhibited by
  interference from Matrix peaks development of
  Naldi
• Spectra VERY dependant upon sample preparation
  and analysis conditions (especially laser power)
  modern instruments have fuzzy logic to
  optimise analytical conditions on the fly

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• Biotechnology applications
• Advances in Proteomics and other areas in
  biotechnology made possible by development of
  soft ionisation Maldi and ESI MS techniques
• Protein and peptide analysis for MW determination
• Protein Identification and profiling using
  digests and data base searching major
  development in Proteomics
• Protein post-translational modification
• Protein structure characterisation
• Maldi-Imaging
• Oligo-nucleotide analysis Confirmation of
  purity of synthetic oligos
• Carbohydrate analysis

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• Biotechnology applications
• Automated high throughput analysis
• Screening of biological samples
• Pharmicokinetics
• LC-MS seperation and identification of
  components of complex mixtures Normally LC-ESI,
  now increasingly LC-Maldi-ToF
• Intact virus analysis
• Cell imaging (Maldi)
• Tissue Imaging (Maldi)

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Mouse Brain Digital Photo Before Matrix Addition
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Mouse Brain HE Stain After Molecular Imaging
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Mouse Brain Full Molecular Spectrum
600-30,000 Da
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Molecular Image of Lipid Mass m/z 786
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Molecular Image of Lipid Mass m/z 1493
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Molecular Image after Unsupervised PCA
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Practical Analytical MS ConsiderationsKnow what
you are trying to achieve Structural
analysis? Accurate Mass Determination?Prepare
sample according to given preparation
protocols Pay attention to sample amount /
concentrationBest results with purified samples
Mixtures of components give reduced spectra
intensity and difficult to identify sample
componentsRemember - you know most about your
sample not the analyst give any and all
available required information.
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Any Questions?