Mass Spectrometry: Methods

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Mass Spectrometry Methods Theory

• David Wishart
• University of Alberta
• Edmonton, AB
• david.wishart_at_ualberta.ca

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MS Principles

• Different elements can be uniquely identified by
  their mass

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MS Principles

• Different compounds can be uniquely identified by
  their mass

Butorphanol L-dopa Ethanol
CH3CH2OH
MW 327.1 MW 197.2 MW 46.1
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Mass Spectrometry

• Analytical method to measure the molecular or
  atomic weight of samples

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Mass Spectrometry

• For small organic molecules the MW can be
  determined to within 5 ppm or 0.0005 which is
  sufficiently accurate to confirm the molecular
  formula from mass alone
• For large biomolecules the MW can be determined
  within an accuracy of 0.01 (i.e. within 5 Da for
  a 50 kD protein)
• Recall 1 dalton 1 atomic mass unit (1 amu)

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Masses in MS

• Monoisotopic mass is the mass determined using
  the masses of the most abundant isotopes
• Average mass is the abundance weighted mass of
  all isotopic components

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Isotopic Distributions
1H 99.9 12C 98.9 35Cl
68.1 2H 0.02 13C 1.1 37Cl
31.9
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Isotopic Distributions
1H 99.9 12C 98.9 35Cl
68.1 2H 0.02 13C 1.1 37Cl
31.9
100
32.1
6.6
2.1
0.06
0.00
m/z
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Mass Calculation (Glycine)
NH2CH2COOH
Amino acid
R1NHCH2COR3
Residue
Glycine Amino Acid Mass 5xH 2xC 2xO 1xN
75.032015 amu Glycine Residue Mass 3xH 2xC
1xO 1xN 57.021455 amu
Monoisotopic Mass 1H 1.007825 12C
12.00000 14N 14.00307 16O 15.99491
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Amino Acid Residue Masses
Monoisotopic Mass
Glycine 57.02147 Alanine 71.03712 Serine 87.03203
Proline 97.05277 Valine 99.06842 Threonine 101.04
768 Cysteine 103.00919 Isoleucine 113.08407 Leucin
e 113.08407 Asparagine 114.04293
Aspartic acid 115.02695 Glutamine 128.05858 Lysin
e 128.09497 Glutamic acid 129.0426 Methionine 13
1.04049 Histidine 137.05891 Phenylalanine 147.068
42 Arginine 156.10112 Tyrosine 163.06333 Tryptop
han 186.07932
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MS History

• JJ Thomson built MS prototype to measure m/z of
  electron, awarded Nobel Prize in 1906
• MS concept first put into practice by Francis
  Aston, a physicist working in Cambridge England
  in 1919
• Designed to measure mass of elements (iso.)
• Aston Awarded Nobel Prize in 1922
• 1920s - Electron impact ionization and magnetic
  sector mass analyzer introduced

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MS History

• 1948-52 - Time of Flight (TOF) mass analyzers
  introduced
• 1955 - Quadrupole ion filters introduced by W.
  Paul, also invents the ion trap in 1983 (wins
  1989 Nobel Prize)
• 1968 - Tandem mass spectrometer appears
• Mass spectrometers are now one of the MOST
  POWERFUL ANALYTIC TOOLS IN CHEMISTRY

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MS Principles

• Find a way to charge an atom or molecule
  (ionization)
• Place charged atom or molecule in a magnetic
  field or subject it to an electric field and
  measure its speed or radius of curvature relative
  to its mass-to-charge ratio (mass analyzer)
• Detect ions using microchannel plate or
  photomultiplier tube

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Mass Spec Principles
Sample

_
Detector
Ionizer
Mass Analyzer
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Typical Mass Spectrometer
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Typical Mass Spectrum
aspirin
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Typical Mass Spectrum

• Characterized by sharp, narrow peaks
• X-axis position indicates the m/z ratio of a
  given ion (for singly charged ions this
  corresponds to the mass of the ion)
• Height of peak indicates the relative abundance
  of a given ion (not reliable for quantitation)
• Peak intensity indicates the ions ability to
  desorb or fly (some fly better than others)

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Resolution Resolving Power

• Width of peak indicates the resolution of the MS
  instrument
• The better the resolution or resolving power, the
  better the instrument and the better the mass
  accuracy
• Resolving power is defined as
• M is the mass number of the observed mass (DM) is
  the difference between two masses that can be
  separated

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Resolution in MS
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Resolution in MS
783.455
QTOF
784.465
785.475
783.6
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Inside a Mass Spectrometer
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Mass Spectrometer Schematic
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Different Ionization Methods

• Electron Impact (EI - Hard method)
• small molecules, 1-1000 Daltons, structure
• Fast Atom Bombardment (FAB Semi-hard)
• peptides, sugars, up to 6000 Daltons
• Electrospray Ionization (ESI - Soft)
• peptides, proteins, up to 200,000 Daltons
• Matrix Assisted Laser Desorption (MALDI-Soft)
• peptides, proteins, DNA, up to 500 kD

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Electron Impact Ionization

• Sample introduced into instrument by heating it
  until it evaporates
• Gas phase sample is bombarded with electrons
  coming from rhenium or tungsten filament (energy
  70 eV)
• Molecule is shattered into fragments (70 eV gtgt
  5 eV bonds)
• Fragments sent to mass analyzer

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EI Fragmentation of CH3OH
CH3OH
CH3OH
CH3OH
CH2OH
H
CH3OH
CH3
OH
CHOH
H
CH2OH
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Why You Cant Use EI For Analyzing Proteins

• EI shatters chemical bonds
• Any given protein contains 20 different amino
  acids
• EI would shatter the protein into not only into
  amino acids but also amino acid sub-fragments and
  even peptides of 2,3,4 amino acids
• Result is 10,000s of different signals from a
  single protein -- too complex to analyze

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Soft Ionization

• Soft ionization techniques keep the molecule of
  interest fully intact
• Electro-spray ionization first conceived in
  1960s by Malcolm Dole but put into practice in
  1980s by John Fenn (Yale)
• MALDI first introduced in 1985 by Franz
  Hillenkamp and Michael Karas (Frankfurt)
• Made it possible to analyze large molecules via
  inexpensive mass analyzers such as quadrupole,
  ion trap and TOF

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Soft Ionization Methods
337 nm UV laser
Fluid (no salt)

_
Gold tip needle
cyano-hydroxy cinnamic acid
MALDI
ESI
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Electrospray (Detail)
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Electrospray (Detail)
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Electrospray Ionization

• Sample dissolved in polar, volatile buffer (no
  salts) and pumped through a stainless steel
  capillary (70 - 150 mm) at a rate of 10-100
  mL/min
• Strong voltage (3-4 kV) applied at tip along with
  flow of nebulizing gas causes the sample to
  nebulize or aerosolize
• Aerosol is directed through regions of higher
  vacuum until droplets evaporate to near atomic
  size (still carrying charges)

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Electrospray Ionization
5H2O/95CH3CN
95H2O/5CH3CN
100 V 1000 V 3000 V
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Electrospray Ionization

• Can be modified to nanospray system with flow lt
  1 mL/min
• Very sensitive technique, requires less than a
  picomole of material
• Strongly affected by salts detergents
• Positive ion mode measures (M H) (add formic
  acid to solvent)
• Negative ion mode measures (M - H)- (add ammonia
  to solvent)

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Positive or Negative Ion Mode?

• If the sample has functional groups that readily
  accept H (such as amide and amino groups found
  in peptides and proteins) then positive ion
  detection is used
• If a sample has functional groups that readily
  lose a proton (such as carboxylic acids and
  hydroxyls as found in nucleic acids and sugars)
  then negative ion detection is used

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Electrospray Ionization

• Samples of MW up to 1200 Da usually produce
  singly charged ions with observed MW equal to
  parent mass H (1.008 Daltons)
• Larger samples (typically peptides) yield ions
  with multiple charges (from 2 to 20 )
• Multiply charged species form a Gaussian
  distribution with those having the most charges
  showing up at lower m/z values

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Multiply Charged Ions
ESI spectrum of HEW Lysozyme MW 14,305.14
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Peptide Masses From ESI
Each peak is given by
m/z mass-to-charge ratio of each peak on
spectrum MW MW of parent molecule n number of
charges (integer) H mass of hydrogen ion
(1.008 Da)
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Peptide Masses From ESI
Charge (n) is unknown, Key is to determine
MW Choose any two peaks separated by 1 charge
1301.4 (MW n1H)
1431.6 (MW nH)
n1
n
2 equations with 2 unknowns - solve for n first
n 1300.4/130.2 10
Substitute 10 into first equation - solve for MW
MW 14316 - (10x1.008) 14305.9
14,305.14
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ESI Transformation

• Software can be used to convert these multiplet
  spectra into single (zero charge) profiles which
  gives MW directly
• This makes MS interpretation much easier and it
  greatly increases signal to noise
• Two methods are available
• Transformation (requires prior peak ID)
• Maximum Entropy (no peak ID required)

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Maximum Entropy
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ESI and Protein Structure

• ESI spectra are actually quite sensitive to the
  conformation of the protein
• Folded, ligated or complexed proteins tend to
  display non-gaussian peak distributions, with few
  observable peaks weighted toward higher m/z
  values
• Denatured or open form proteins/peptides which
  ionize easier tend to display many peaks with a
  classic gaussian distribution

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ESI and Protein Conformation
Native Azurin
Denatured Azurin
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Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy cinnamic acid
MALDI
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MALDI

• Sample is ionized by bombarding sample with laser
  light
• Sample is mixed with a UV absorbant matrix
  (sinapinic acid for proteins, 4-hydroxycinnaminic
  acid for peptides)
• Light wavelength matches that of absorbance
  maximum of matrix so that the matrix transfers
  some of its energy to the analyte (leads to ion
  sputtering)

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MALDI Ionization
Matrix

• Absorption of UV radiation by chromophoric matrix
  and ionization of matrix
• Dissociation of matrix, phase change to
  super-compressed gas, charge transfer to analyte
  molecule
• Expansion of matrix at supersonic velocity,
  analyte trapped in expanding matrix plume
  (explosion/popping)

-
-
Laser
-

Analyte



-

-

-

-






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MALDI

• Unlike ESI, MALDI generates spectra that have
  just a singly charged ion
• Positive mode generates ions of M H
• Negative mode generates ions of M - H
• Generally more robust that ESI (tolerates salts
  and nonvolatile components)
• Easier to use and maintain, capable of higher
  throughput
• Requires 10 mL of 1 pmol/mL sample

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MALDI Sample Limits

• Phosphate buffer lt 50 mM
• Ammonium bicarbonate lt 30 mM
• Tris buffer lt 100 mM
• Guanidine (chloride, sulfate) lt 1 M
• Triton lt 0.1
• SDS lt 0.01
• Alkali metal salts lt 1 M
• Glycerol lt 1

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MALDI SELDI
337 nm UV laser
cyano-hydroxy cinnaminic acid
MALDI
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MALDI/SELDI Spectra
Normal
Tumor
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Mass Spectrometer Schematic
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Different Mass Analyzers

• Magnetic Sector Analyzer (MSA)
• High resolution, exact mass, original MA
• Quadrupole Analyzer (Q)
• Low (1 amu) resolution, fast, cheap
• Time-of-Flight Analyzer (TOF)
• No upper m/z limit, high throughput
• Ion Trap Mass Analyzer (QSTAR)
• Good resolution, all-in-one mass analyzer
• Ion Cyclotron Resonance (FT-ICR)
• Highest resolution, exact mass, costly

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Magnetic Sector Analyzer
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Mass Spec Equation (Magnet Sector)
B2 r2
m

z
2V
M mass of ion B magnetic field z charge of
ion r radius of circle V voltage
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Quadrupole Mass Analyzer
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Quadrupole Mass Analyzer

• A quadrupole mass filter consists of four
  parallel metal rods with different charges
• Two opposite rods have an applied potential of
  (UVcos(wt)) and the other two rods have a
  potential of -(UVcos(wt))
• The applied voltages affect the trajectory of
  ions traveling down the flight path
• For given dc and ac voltages, only ions of a
  certain mass-to-charge ratio pass through the
  quadrupole filter and all other ions are thrown
  out of their original path

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Q-TOF Mass Analyzer
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Mass Spec Equation (TOF)
2Vt2
m

z
L2
m mass of ion L drift tube length z charge
of ion t time of travel V voltage
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Ion Trap Mass Analyzer

• Ion traps are ion trapping devices that make use
  of a three-dimensional quadrupole field to trap
  and mass-analyze ions
• invented by Wolfgang Paul (Nobel Prize1989)
• Offer good mass resolving power, and even MSn
  capability.

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Ion Trap Mass Analyzer
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FT-Ion Cyclotron Analzyer
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FT-ICR

• Uses powerful magnet (5-10 Tesla) to create
  miniature cyclotron
• Originally developed in Canada (UBC) by A.G.
  Marshal in 1974
• FT approach allows many ion masses to be
  determined simultaneously (efficient)
• Has higher mass resolution than any other MS
  analyzer available
• Will revolutionize proteomics studies

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Mass Spectrometer Schematic
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MS Detectors

• Early detectors used photographic film
• Todays detectors (ion channel and electron
  multipliers) produce electronic signals via 2o
  electronic emission when struck by an ion
• Timing mechanisms integrate these signals with
  scanning voltages to allow the instrument to
  report which m/z has struck the detector
• Need constant and regular calibration

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Mass Detectors
Electron Multiplier (Dynode)
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Different Types of MS

• ESI-QTOF
• Electrospray ionization source quadrupole mass
  filter time-of-flight mass analyzer
• MALDI-QTOF
• Matrix-assisted laser desorption ionization
  quadrupole time-of-flight mass analyzer

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Different Types of MS

• GC-MS - Gas Chromatography MS
• separates volatile compounds in gas column and
  IDs by mass
• LC-MS - Liquid Chromatography MS
• separates delicate compounds in HPLC column and
  IDs by mass
• MS-MS - Tandem Mass Spectrometry
• separates compound fragments by magnetic field
  and IDs by mass

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Tandem Mass Spectrometer
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Tandem Mass Spectrometry

• Purpose is to fragment ions from parent ion to
  provide structural information about a molecule
• Also allows separation and identification of
  compounds in complex mixtures
• Uses two or more mass analyzers/filters separated
  by a collision cell filled with Argon or Xenon
• Collision cell is where selected ions are sent
  for further fragmentation

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Tandem Mass Spectrometry

• Different MS-MS configurations
• Quadrupole-quadrupole (low energy)
• Magnetic sector-quadrupole (high)
• Quadrupole-time-of-flight (low energy)
• Time-of-flight-time-of-flight (low energy)
• Fragmentation experiments may also be performed
  on single analyzer instruments such as ion trap
  instruments and TOF instruments equipped with
  post-source decay

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Different MS-MS Modes

• Product or Daughter Ion Scanning
• first analyzer selects ion for further
  fragmentation
• most often used for peptide sequencing
• Precursor or Parent Ion Scanning
• no first filtering, used for glycosylation
  studies
• Neutral Loss Scanning
• selects for ions of one chemical type (COOH, OH)
• Selected/Multiple Reaction Monitoring
• selects for known, well characterized ions only

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MS-MS Proteomics
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Proteomics Applications

• Protein sample identification/confirmation
• Protein sample purity determination
• Detection of post-translational modifications
• Detection of amino acid substitutions
• Determination of disulfide bonds ( status)
• De novo peptide sequencing
• Mass fingerprint identification of proteins
• Monitoring protein folding (H/D exchange)
• Monitoring protein-ligand complexes/struct.

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Conclusions

• Mass spectrometers exist in many different
  configurations to allow different problems to be
  solved
• All mass spectrometers have a common architecture
  and relatively similar operating principles
• Understanding the applications and limitations of
  MS in proteomics will help in understanding and
  meeting the bioinformatics needs in proteomics