Mass Spectrometry:
Methods & Theory
MS Principles
• Different elements can be uniquely
identified by their mass
2
MS Principles
• Different compounds can be uniquely
identified by their mass
Butorphanol
L-dopa
N -CH2OH
Ethanol
COOH
HO
-CH2CH-NH2
CH3CH2OH
HO
HO
MW = 327.1
MW = 197.2
MW = 46.1
3
Mass Spectrometry
• Analytical method to measure the
molecular or atomic weight of samples
4
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)
5
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
6
Isotopic Distributions
1H
= 99.9%
2H = 0.02%
12C
= 98.9%
13C = 1.1%
35Cl
= 68.1%
37Cl = 31.9%
7
Isotopic Distributions
1H
12C
= 99.9%
2H = 0.02%
= 98.9%
13C = 1.1%
35Cl
= 68.1%
37Cl = 31.9%
100
32.1
6.6
2.1
0.06 0.00
m/z
8
Mass Calculation (Glycine)
NH2—CH2—COOH
Amino acid
R1—NH—CH2—CO—R3
Residue
Monoisotopic Mass
1H = 1.007825
12C = 12.00000
14N = 14.00307
16O = 15.99491
Glycine Amino Acid Mass
5xH + 2xC + 2xO + 1xN
= 75.032015 amu
Glycine Residue Mass
3xH + 2xC + 1xO + 1xN
=57.021455 amu
9
Amino Acid Residue Masses
Monoisotopic Mass
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
57.02147
71.03712
87.03203
97.05277
99.06842
101.04768
103.00919
113.08407
113.08407
114.04293
Aspartic acid
Glutamine
Lysine
Glutamic acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan
115.02695
128.05858
128.09497
129.0426
131.04049
137.05891
147.06842
156.10112
163.06333
186.07932
10
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
11
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
12
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
13
Mass Spec Principles
Sample
+
_
Ionizer
Mass Analyzer
Detector
14
Typical Mass Spectrometer
15
Typical Mass Spectrum
aspirin
16
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 ion’s ability to
desorb or “fly” (some fly better than others)
17
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
DM
• Resolving power is defined as:
M
• M is the mass number of the observed mass
(DM) is the difference between two masses
that can be separated
18
Resolution in MS
19
Resolution in MS
783.455
QTOF
784.465
785.475
783.6
20
Inside a Mass Spectrometer
21
Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Sample Plate
Target
HPLC
GC
Solids probe
Ion
Source
Mass
Filter
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Detector
Microch plate
Electron Mult.
Hybrid Detec.
Data
System
PC’s
UNIX
Mac
22
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
23
24
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 >> 5 eV bonds)
• Fragments sent to mass analyzer
25
EI Fragmentation of CH3OH
CH3OH
CH3OH+
CH3OH
CH2O=H+
CH3OH
+
CH2O=H+
+ H
CH3 + OH
CHO=H+ + H
26
Why You Can’t 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 subfragments and even peptides of 2,3,4…
amino acids
• Result is 10,000’s of different signals from
a single protein -- too complex to analyze
27
Soft Ionization
• Soft ionization techniques keep the
molecule of interest fully intact
• Electro-spray ionization first conceived in
1960’s by Malcolm Dole but put into
practice in 1980’s 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
28
29
Soft Ionization Methods
337 nm UV laser
Fluid (no salt)
+
_
cyano-hydroxy
cinnamic acid
Gold tip needle
MALDI
ESI
30
Electrospray (Detail)
31
Electrospray (Detail)
32
Electrospray Ionization
• Sample dissolved in polar, volatile buffer
(no salts) and pumped through a stainless
steel capillary (70 - 150 mm) at a rate of 10100 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)
33
Electrospray Ionization
5%H2O/95%CH3CN
95%H2O/5%CH3CN
100 V
1000 V
3000 V
34
Electrospray Ionization
• Can be modified to “nanospray” system
with flow < 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)
35
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
36
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
37
Multiply Charged Ions
ESI spectrum of
HEW Lysozyme
MW = 14,305.14
38
Peptide Masses From ESI
Each peak is given by:
m/z = (MW + nH+)
n
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)
39
Peptide Masses From ESI
Charge (n) is unknown, Key is to determine MW
Choose any two peaks separated by 1 charge
1431.6 = (MW + nH+) 1301.4 = (MW + [n+1]H+)
[n+1]
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
40
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)
41
Maximum Entropy
42
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
43
ESI and Protein Conformation
Native Azurin
Denatured Azurin
44
Matrix-Assisted Laser
Desorption Ionization
337 nm UV laser
cyano-hydroxy
cinnamic acid
MALDI
45
MALDI
• Sample is ionized by bombarding sample
with laser light
• Sample is mixed with a UV absorbant
matrix (sinapinic acid for proteins, 4hydroxycinnaminic 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)
46
MALDI Ionization
Matrix
+
+ +-+
Laser
Analyte
+
+ ++ + --+
-+
+
+
+
+
+
• Absorption of UV radiation
by chromophoric matrix and
ionization of matrix
• Dissociation of matrix,
phase change to supercompressed gas, charge
transfer to analyte molecule
• Expansion of matrix at
supersonic velocity, analyte
trapped in expanding matrix
plume (explosion/”popping”)
47
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
48
MALDI Sample Limits
•
•
•
•
•
•
•
•
Phosphate buffer < 50 mM
Ammonium bicarbonate < 30 mM
Tris buffer < 100 mM
Guanidine (chloride, sulfate) < 1 M
Triton < 0.1%
SDS < 0.01%
Alkali metal salts < 1 M
Glycerol < 1%
49
MALDI = SELDI
337 nm UV laser
cyano-hydroxy
cinnaminic acid
MALDI
50
MALDI/SELDI Spectra
Normal
Tumor
51
Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Sample Plate
Target
HPLC
GC
Solids probe
Ion
Source
Mass
Filter
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Detector
Microch plate
Electron Mult.
Hybrid Detec.
Data
System
PC’s
UNIX
Mac
52
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
53
Magnetic Sector Analyzer
54
Mass Spec Equation
(Magnet Sector)
2
2
B
r
m
=
z
2V
M = mass of ion
z = charge of ion
V = voltage
B = magnetic field
r = radius of circle
55
Quadrupole Mass Analyzer
56
Quadrupole Mass Analyzer
• A quadrupole mass filter consists of four
parallel metal rods with different charges
• Two opposite rods have an applied
potential of (U+Vcos(wt)) and the other two
rods have a potential of -(U+Vcos(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
57
Q-TOF Mass Analyzer
NANOSPRAY
TIP
MCP
DETECTOR
PUSHER
HEXAPOLE
QUADRUPOLE
ION
SOURCE
HEXAPOLE
COLLISION
CELL
TOF
REFLECTRON
SKIMMER
HEXAPOLE
58
Mass Spec Equation (TOF)
2
2Vt
m
=
z
L2
m = mass of ion
z = charge of ion
V = voltage
L = drift tube length
t = time of travel
59
Ion Trap Mass Analyzer
• Ion traps are ion
trapping devices that
make use of a threedimensional quadrupole
field to trap and massanalyze ions
• invented by Wolfgang
Paul (Nobel Prize1989)
• Offer good mass
resolving power, and
even MSn capability.
60
Ion Trap Mass Analyzer
61
FT-Ion Cyclotron Analzyer
62
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
63
Mass Spectrometer Schematic
Turbo pumps
Diffusion pumps
Rough pumps
Rotary pumps
High Vacuum System
Inlet
Sample Plate
Target
HPLC
GC
Solids probe
Ion
Source
Mass
Filter
MALDI
ESI
IonSpray
FAB
LSIMS
EI/CI
TOF
Quadrupole
Ion Trap
Mag. Sector
FTMS
Detector
Microch plate
Electron Mult.
Hybrid Detec.
Data
System
PC’s
UNIX
Mac
64
MS Detectors
• Early detectors used photographic film
• Today’s 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
65
Mass Detectors
Electron Multiplier (Dynode)
66
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
67
Different Types of MS
• GC-MS - Gas Chromatography MS
– separates volatile compounds in gas column
and ID’s by mass
• LC-MS - Liquid Chromatography MS
– separates delicate compounds in HPLC
column and ID’s by mass
• MS-MS - Tandem Mass Spectrometry
– separates compound fragments by magnetic
field and ID’s by mass
68
Tandem Mass Spectrometer
NANOSPRAY
TIP
MCP
DETECTOR
PUSHER
HEXAPOLE
QUADRUPOLE
ION
SOURCE
HEXAPOLE
COLLISION
CELL
TOF
REFLECTRON
SKIMMER
HEXAPOLE
69
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
70
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
71
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
72
MS-MS & Proteomics
73
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.
74
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
75