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Mass Spectrometry: Methods

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Mass Spectrometry: Methods & Theory David Wishart University of Alberta Edmonton, AB david.wishart_at_ualberta.ca – PowerPoint PPT presentation

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Title: Mass Spectrometry: Methods


1
Mass Spectrometry Methods Theory
  • David Wishart
  • University of Alberta
  • Edmonton, AB
  • david.wishart_at_ualberta.ca

2
MS Principles
  • Different elements can be uniquely identified by
    their mass

3
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
4
Mass Spectrometry
  • Analytical method to measure the molecular or
    atomic weight of samples

5
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)

6
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

7
Isotopic Distributions
1H 99.9 12C 98.9 35Cl
68.1 2H 0.02 13C 1.1 37Cl
31.9
8
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
9
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
10
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
11
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

12
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

13
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

14
Mass Spec Principles
Sample

_
Detector
Ionizer
Mass Analyzer
15
Typical Mass Spectrometer
16
Typical Mass Spectrum
aspirin
17
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)

18
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

19
Resolution in MS
20
Resolution in MS
783.455
QTOF
784.465
785.475
783.6
21
Inside a Mass Spectrometer
22
Mass Spectrometer Schematic
23
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

24
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25
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

26
EI Fragmentation of CH3OH
CH3OH
CH3OH
CH3OH
CH2OH
H
CH3OH
CH3
OH
CHOH
H
CH2OH
27
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

28
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

29
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30
Soft Ionization Methods
337 nm UV laser
Fluid (no salt)

_
Gold tip needle
cyano-hydroxy cinnamic acid
MALDI
ESI
31
Electrospray (Detail)
32
Electrospray (Detail)
33
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)

34
Electrospray Ionization
5H2O/95CH3CN
95H2O/5CH3CN
100 V 1000 V 3000 V
35
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)

36
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

37
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

38
Multiply Charged Ions
ESI spectrum of HEW Lysozyme MW 14,305.14
39
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)
40
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
41
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)

42
Maximum Entropy
43
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

44
ESI and Protein Conformation
Native Azurin
Denatured Azurin
45
Matrix-Assisted Laser Desorption Ionization
337 nm UV laser
cyano-hydroxy cinnamic acid
MALDI
46
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)

47
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



-

-

-

-






48
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

49
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

50
MALDI SELDI
337 nm UV laser
cyano-hydroxy cinnaminic acid
MALDI
51
MALDI/SELDI Spectra
Normal
Tumor
52
Mass Spectrometer Schematic
53
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

54
Magnetic Sector Analyzer
55
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
56
Quadrupole Mass Analyzer
57
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

58
Q-TOF Mass Analyzer
59
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
60
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.

61
Ion Trap Mass Analyzer
62
FT-Ion Cyclotron Analzyer
63
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

64
Mass Spectrometer Schematic
65
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

66
Mass Detectors
Electron Multiplier (Dynode)
67
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

68
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

69
Tandem Mass Spectrometer
70
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

71
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

72
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

73
MS-MS Proteomics
74
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.

75
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
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