Transcript Mass Spectrometry - MITCON Biopharma

Mass Spectrometry
Definition of Mass Spectrometry
Mass spectrometry (MS) :
An analytical technique by using mass spectrometry
for the determination of the composition of a sample
or molecule and elucidation of the chemical
structures of molecules, such as peptides and other
chemical compounds.
Mass spectrometry has been described as the
smallest scale in the world, not because of the mass
spectrometer’s size but because of the size of what it
weighs -- molecules.
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-tocharge 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)
• m=molecular or atomic mass, z = electrostatic charge unit
• In many cases (such as small molecules), z = 1
 measured m/z = mass of fragment
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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:
1. Sample Inlet
2. Ion Source
3. Mass Analyzer
•
Detector
•
Data System
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How does it work?
accelerate
ionise
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Magnetic and/or
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vapourise
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heavy
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light
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Mass spectrometry
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Mass Spectrometer
=>
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
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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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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Ionization Methods
• Electron bomb Ionization (EI
• Chemical Ionization ( CI
• Field ionization () FI
•
Matrix Assisted Laser Desorption Ionization ( MALDI
•
Fast atom bombardment ) FAB
•
Electro Spray Ionization () ESI
Electron Bomb Ionization ( EI )
Sample is heated and energized by a beam of electrons, usually gives a
molecular ion (M+) and a lot of fragments。
H H
H C C H
H H
H H
e- +
H H
H C C H
H C C+
H H
H H
H
H C+
H
H
C H
H
H
Electron Bomb Ionization ( EI )
Properties of EI
Hard ionization
Gas-phase molecules enter source through heated probe or
GC column
70 eV electrons bombard molecules forming M+* ions that
fragment in unique reproducible way to form a collection
of fragment ions
EI spectra can be matched to library stds CI (soft ionization)
Higher pressure of methane leaked into the source (mtorr)
Reagent ions transfer proton to analyte
Chemical Ionization (CI)
Electron ionization leads to fragmentation of the
molecular ion, which sometimes prevents its detection.
Chemical ionization (CI):
A technique that produces ions with little excess energy.
Thus this technique presents the advantage of yielding a
spectrum with less fragmentation in which the molecular
species is easily recognized.
Consequently, chemical ionization is complementary to
electron ionization.
Chemical Ionization (CI)
Properties of CI
Advantages
Parent Ion
Interface to GC
Insoluble Samples
Disadvantages
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds
(<1000 amu)
Solids Probe Requires
Skilled Operator
Field ionization (FI)
Field ionization (FI) is a method that uses very strong electric
fields to produce ions from gas-phase molecules.
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d<1mm
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Field ionization (FI)
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- + - +
+ - + + - ++ +
+ +
- + + - +
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Matrix Assisted Laser Desorption
Ionization (MALDI)
sample is co-crystallized with a matrix and then irradiated
with laser.
MALDI is achieved in two steps. In the first step, the
compound to be analyzed is dissolved in a solvent
containing in solution small organic molecules, called
the matrix. The second step occurs under vacuum
conditions inside the source of the mass spectrometer.
Properties of MALDI
Good solubility
Vapour pressure must be sufficiently low to maintain vacuum conditions
Viscosity must allow diffusion of the analyte from the bulk to the surface
Polar : to solvate and separate preformed ion
Less Sensitive to Salts
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)
Principle of MALDI
MALDI mass spectrometry has become a powerful analytical
tool for both synthetic polymers and biopolymers.
Fast atom bombardment ( FAB)
Softer than EI and CI. Ions are produced by bombardment
with heavy atoms. Gives (M+H)+ ions and litle fragmentation.
Good for more polar compounds.
Ar + e
Ar+ + Ar
fast
slow
Ar+
acceleration (5-15 KeV)
Ar + Ar+
+ 8 KeV
fast
slow
Properties of FAB
Advantages
Parent Ion
High Mass Compounds
(10,000 amu)
Thermally Labile
Compounds (R.T.)
Disadvantages
No Fragment Library
Solubility in Matrix
(MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled
Operator
Relatively Low Sensitivity
ElectroSpray Ionization (ESI)
Electrospray is abbreviated to ESI ，ample is sprayed out of
a narrow nozzle in a high potential field. Generates positive
(M+nH)n+ and negative (M - nH)n- ions and almost no
fragmentation. Generates multiple charged ions.
2. Principle
Properties of ESI
Advantages
Electrospray Ionization can
be
easily interfaced to LC.
Absolute signals from
Electrospray are more easily
reproduced, therefore, better
quantitation.
Mass Accuracy is considered
better.
Multiple charging is more
common then MALDI.
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar
Solvent (MeOH, ACN,
H2O, Acetone are best)
Sensitive to Salts
Suppression
Types of Mass Analyzers
Magnetic sector analyzer
Time of Flight analyzer (TOF)
Quadrupole analyzers
Fourier Transform Ion-Cyclotron
Magnetic Sector Analyzer
Magnetic sector analyzer – Uses electric and/or
magnetic fields to separate ions
Principle of Magnetic Sector
Analyzer
The ion source accelerates ions to a kinetic
energy given by : (1/2)m2= zV
Where m is the mass of the ion ,v is its velocity, z
is the charge on the ion ,and V is the applied
voltage of the ion optics.
Principle of Magnetic Sector
Analyzer
Only ions of mass-to-charge ratio that have equal
centripetal and centrifugal forces pass through the
flight tube
It shows that the m/q ratio of the ions that reach
the detector can be varied by changing either the
magnetic field or the applied voltage of the ion
optics.
In summary ,by varying the voltage or magnetic
field of the magnetic-sector analyzer ,the individual
ion beams are separated spatially and each has a
unique radius of curvature according to its
mass/charge ratio.
Advantages
Double focusing magnetic sector mass analyzers are the
"classical" model against which other mass analyzers are
compared.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all MS analyzers
High resolution
High sensitivity
10,000 Mass Range
Linked scan MS/MS does not require another analyzer

Disadvantages
Requires Skilled Operator
Usually larger and higher cost than other mass analyzers
Difficult to interface to ESI
Low resolution MS/MS without multiple analyzers

Applications
All organic MS analysis methods
Accurate mass measurements
Quantitation
Isotope ratio measurements
Time of Flight Analyzer
TOF analyzer – ions are accelerated through a flight tube
and the time of light to the detector is measured
Ions are accelerated and their time of flight to the
detector is measured.
Principle of TOF Analyzer

Uses a pulse of ion mixtures, not steady stream

Ions accelerated into drift tube by a pulsed electric

field called the ion-extraction field

Drift Tube is usually 1-2 m long, under vacuum

Ions traverse the drift tube at different speeds

( L / t ) = v = ( 2zV / m )½
Advantages of TOF Analyzer
Good for kinetic studies of fast reactions and for
use with gas chromatography to analyze peaks
from chromatograph
High ion transmission
Can register molecular ions that decompose in the
flight tube
Extremely high mass range (>1MDa)
Fastest scanning

Disadvantages
Requires pulsed ionization method or ion beam
switching (duty cycle is a factor)
Low resolution (4000)
Limited precursor-ion selectivity for most MS/MS
experiments

Applications
Almost all MALDI systems
Very fast GC/MS systems
Quadrupole Analyzers
Quadrupole analyzers – ions
are filtered or trapped in a
device consisting of several
metal rods using specifically
tailored electromagnetic
fields
Quadrupole Analyzers
• Electric/magnetic fields trap, store, eject ions
• Requires an in-line quadrupole to act as
mass pre-filter
• Contains a single ring electrode and a top
and bottom cap electrode
• Varying RF frequency will vary the m/z ratios
that are trapped
• Additional fragmentation can be performed
on ions stored in the ion trap

Advantages
Easy to use ,simple construction,fast
Good reproducibility
Relatively small and low-cost systems
Quadrupoles are now capable of routinely
analyzing up to a m/q ratio of 3000,which is
useful in electrospary ionization of biomolecules,
which commonly produces a charge distribution
below m/z 3000

Disadvantages
Low resolution(<4000)
Slow scanning
Low accuracy (>100ppm)

Applications
Majority of benchtop GC/MS and LC/MS systems
Separation of proteins and other biomolecules
with electrosprary
Sector / quadrupole hybrid MS/MS systems
Fourier Transform Ion Cyclotron
Resonance (FT ICR) analyzers
Most FTICR mass spectrometers use superconducting
magnets, which provide a relatively stable calibration over a
long period of time.
Although some mass accuracy can be obtained without
internal calibrant, mass accuracy and resolution are inversely
proportional to m/z, and the best accurate mass measurements
require an internal calibrant.
Unlike the quadrupole ion trap, the FTICR mass spectrometer
is not operated as a scanning device.

Advantages
The highest recorded mass resolution of all mass
spectrometers (>500,000)
Very good accuracy (<1ppm)
Well-suited for use with pulsed ionization
methods such as MALDI
Non-destructive ion detection; ion remeasurement
Stable mass calibration in superconducting
magnet FTICR systems

Disadvantages
Expensive
Requires superconducting magnet
Subject to space charge effects and ion molecule reactions
Artifacts such as harmonics and sidebands are present in the
mass spectra
Many parameters (excitation, trapping, detection conditions)
comprise the experiment sequence that defines the quality of the
mass spectrum
Generally low-energy CID, spectrum depends on collision
energy, collision gas, and other parameters.

Applications
Ion chemistry
High-resolution MALDI and electrospray
experiments for high-mass analytes
Laser desorption for materials and surface
characterizatio
The Mass Spectrum
A. Presentation of data
1. The mass spectrum is presented in terms of ion abundance vs.
m/e ratio (mass).
2. The most abundant ion formed in ionization gives rise to the
tallest peak on the mass spectrum – this is the base peak.
base peak, m/e 43
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A. Presentation of data
3. All other peak intensities are relative to the base peak as a
percentage.
4. If a molecule loses only one electron in the ionization process, a
molecular ion is observed that gives its molecular weight – this
is designated as M+ on the spectrum.
M+, m/e 114
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A. Presentation of data
5. In most cases, when a molecule loses a valence electron, bonds
are broken, or the ion formed quickly fragment to lower
energy ions
6. The masses of charged ions are recorded as fragment ions by
the spectrometer – neutral fragments are not recorded !
fragment ions
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B. Determination of Molecular Mass
1. When a M+ peak is observed it gives the molecular mass –
assuming that every atom is in its most abundant isotopic form
2. Remember that carbon is a mixture of 98.9% 12C (mass 12),
1.1% 13C (mass 13) and <0.1% 14C (mass 14)
3. We look at a periodic table and see the atomic weight of
carbon as 12.011 – an average molecular weight
4. The mass spectrometer, by its very nature would see a peak at
mass 12 for atomic carbon and a M + 1 peak at 13 that would
be 1.1% as high
- We will discuss the effects of this later…
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B. Determination of Molecular Mass
5.The Nitrogen Rule is another means of confirming the
observance of a molecular ion peak
6. If a molecule contains an even number of nitrogen atoms (only
‘common’ organic atom with an odd valence) or no nitrogen
atoms the molecular ion will have an even mass value
7. If a molecule contains an odd number of nitrogen atoms, the
molecular ion will have an odd mass value
8. If the molecule contains chlorine or bromine, each with two
common isotopes, the determination of M+ can be made
much easier, or much more complex as we will see.
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