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Mass Spectrometry:
Ionization methods
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Mass Spec Principles
Ionizer
Sample
+
_
Mass Analyzer Detector
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How does a mass spectrometer work?
Ionization
method
MALDI
Electrospray
Mass analyzer
MALDI-TOF
MW
Triple Quadrapole AA seq
MALDI-QqTOF AA seq and MW
QqTOF AA seq and protein modif.
Create ions Separate ions Detect ions
Mass
spectrum
Database
analysis
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Ion sources for molecular mass
spectrometry
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Soft Ionization Soft ionization techniques keep the molecule
of interest fully intact
Electro-spray ionization first conceived in1960s 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 moleculesvia inexpensive mass analyzers such as
quadrupole, ion trap and TOF
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Soft Ionization Methods
337 nm UV laser
MALDI
cyano-hydroxy
cinnamic acidGold tip needle
Fluid (no salt)
ESI
+
_
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Soft ion sources little excess energy in
molecule
reduced fragmentation
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Hard ion sources leave excess energy in
molecule-extensive fragmentation
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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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Sample introduction/ionization method
Ionization
method
Typical
Analytes
Sample
Introduction
Mass
Range
Method
Highlights
Electron Impact (EI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Hard method
versatile
provides
structure info
Chemical Ionization (CI)
Relatively
smallvolatile
GC or
liquid/solidprobe
to
1,000Daltons
Soft method
molecular ionpeak [M+H]+
Electrospray (ESI)
Peptides
Proteins
nonvolatile
Liquid
Chromatography
or syringe
to
200,000
Daltons
Soft method
ions often
multiply
charged
Fast Atom Bombardment
(FAB)
Carbohydrates
Organometallics
Peptides
nonvolatile
Sample mixed
in viscous
matrix
to
6,000
Daltons
Soft method
but harder
than ESI or
MALDI
Matrix Assisted Laser
Desorption
(MALDI)
Peptides
Proteins
Nucleotides
Sample mixed
in solid
matrix
to
500,000
Daltons
Soft method
very high
mass
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Electron Ionization
Also called as electron impact ionization
Oldest and best-characterized of all the ionization
methods.
A beam of electrons passes through the gas-phase
sample. An electron that collides with a neutral analyte
molecule can knock off another electron, resulting in a
positively charged ion.
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Electron Ionization
The ionization process can either produce a molecular
ion which will have the same molecular weight andelemental composition of the starting analyte, or it can
produce a fragmention which corresponds to a smaller
piece of the analyte molecule.
Most mass spectrometers use electrons with anenergy of 70 electron volts (eV) for EI.
Decreasing the electron energy can reduce
fragmentation, but it also reduces the number of ionsformed.
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EI Fragmentation of CH3OH
CH3OH CH3OH+
CH3OH CH2O=H+ + H
CH3OH+ CH3 + OH
CHO=H+ + HCH2O=H+
Why wouldnt Electron Impact be suitable
for analyzing proteins?
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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 onlyinto amino acids but also amino acid sub-
fragments and even peptides of 2,3,4amino acids
Result is 10,000s of different signals from
a single protein -- too complex to analyze
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Electron Ionization
Sample introduction
heated batch inlet
heated direct insertion probe
gas chromatography
liquid chromatography (particle-beaminterface)
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Some typical reactions in EI
source
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Fragmentation patterns
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Fragmentation patterns
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Electron Ionization
Benefits well-understood
can be applied to virtually all volatile
compounds reproducible mass spectra
fragmentation provides structural information
libraries of mass spectra can be searched forEI mass spectral "fingerprint"
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Electron Ionization
Limitations
sample must be thermally volatile and
stable
the molecular ion may be weak orabsent for many compounds.
Mass range
Low Typically less than 1,000 Da.
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Electron Ionization
Advantages of EI: high ion currents - sensitive
fragmentation aids identification
Disadvantages of EI:
weak or absent M+ peak inhibitsdetermination of MW
molecules must be vaporized (MW < 103 Da)
molecules must be thermally stable duringvaporization
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Chemical Ionization (CI) Chemical ionization uses ion-molecule reactions to
produce ions from the analyte. The chemical ionization process begins when a
reagent gas such as methane, isobutane, or
ammonia is ionized by electron impact.
A high reagent gas pressure (or long reaction time)
results in ion-molecule reactions between the
reagent gas ions and reagent gas neutrals.
Some of the products of these ion-moleculereactions can react with the analyte molecules to
produce analyte ions.
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Chemical Ionization (CI)Example (R = reagent, S = sample, e = electron, . =
radical electron , H = hydrogen):R + e ---> R+. + 2e
R+. + RH ---> RH+ + R.
RH+ + S ---> SH+ + R(of course, other reactions can occur)
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Chemical Ionization (CI)Sample introduction
heated batch inlet heated direct insertion probe
gas chromatograph
liquid chromatograph (particle-beam interface)Benefits
often gives molecular weight information through
molecular-like ions such as [M+H]+, even when EIwould not produce a molecular ion.
simple mass spectra, fragmentation reducedcompared to EI
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Chemical Ionization (CI)Limitations
sample must be thermally volatile and stable less fragmentation than EI, fragment pattern not
informative or reproducible enough for library
search results depend on reagent gas type, reagent gas
pressure or reaction time, and nature of sample.
Mass range
Low Typically less than 1,000 Da.
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Field Desorption (FD)
The sample is deposited onto the emitter and the emitter is
biased to a high potential (several kilovolts) and a current is
passed through the emitter to heat up the filament.
Mass spectra are acquired as the emitter current is
gradually increased and the sample is evaporated from the
emitter into the gas phase.
The analyte molecules are ionized by electron tunneling atthe tip of the emitter 'whiskers'.
Characteristic positive ions produced are radical molecular
ions and cation attached species such as [M+Na]+ and [M-
Na]+. The latter are probably produced during desorption by the
attachment of trace alkali metal ions present in the analyte.
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Field Desorption (FD)
Sample introduction
Direct insertion probe. The sample is deposited onto the tip of the emitter
by
. dipping the emitter into an analyte solution
. depositing the dissolved or suspended sample
onto the emitter with a microsyringe
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Field Desorption (FD)
Benefits
simple mass spectra, typically one molecular ormolecular-like ionic species per compound.
little or no chemical background
works well for small organic molecules, manyorganometallics, low molecular weight polymersand some petrochemical fractions
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Field Desorption (FD)
Limitations
sensitive to alkali metal contamination and sampleoverloading
emitter is relatively fragile
relatively slow analysis as the emitter current is
increased the sample must be thermally volatile to some
extent to be desorbed
Mass range Low-moderate, depends on the sample. Typically
less than about 2,000 to 3,000 Da.
some examples have been recorded from ions withmasses beyond 10,000 Da.
Fi ld I i ti (FI)
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Field Ionization (FI)
The sample is evaporated from a direct insertion probe,gas chromatograph, or gas inlet.
As the gas molecules pass near the emitter, they areionized by electron tunneling.
Sample introduction heated direct insertion probe
gas inlet
gas chromatograph
i i i
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Field Ionization (FI)
Benefits
simple mass spectra, typically one molecular ormolecular-like ionic species per compound.
little or no chemical background
works well for small organic molecules and somepetrochemical fractions
Fi ld I i i (FI)
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Field Ionization (FI)
Limitations
The sample must be thermally volatile.
Samples are introduced in the same way as forelectron ionization (EI).
Mass range
Low Typically less than 1000 Da.
F t At B b d t (FAB)
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Fast Atom Bombardment (FAB)
The analyte is dissolved in a liquid matrix such as
glycerol, thioglycerol,m-nitrobenzyl alcohol,
ordiethanolamine and a small amount (about 1microliter) is placed on a target.
The target is bombarded with a fast atom beam
(for example, 6 keV xenon atoms) that desorbmolecular-like ions and fragments from theanalyte.
Cluster ions from the liquid matrix are alsodesorbed and produce a chemical background thatvaries with the matrix used.
F t At B b d t (FAB)
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Fast Atom Bombardment (FAB)
Sample introduction
direct insertion probe LC/MS (frit FAB or continuous-flow FAB).
Benefits rapid
simple
relatively tolerant of variations in sampling
good for a large variety of compounds
strong ion currents -- good for high-resolutionmeasurements
F t At B b d t (FAB)
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Fast Atom Bombardment (FAB)
Limitations
high chemical background defines detection limits
may be difficult to distinguish low-molecular-weightcompounds from chemical background
analyte must be soluble in the liquid matrix
no good for multiply charged compounds with morethan 2 charges
Mass range
Moderate Typically ~300 Da to about 6000 Da.
Secondar Ion Mass Spectrometr (SIMS)
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Secondary Ion Mass Spectrometry (SIMS)
Dynamic SIMS is nearly identical to FAB except
that the primary particle beam is an ion beam(usually cesium ions) rather than a neutral beam.
The ions can be focused and accelerated to higher
kinetic energies than are possible for neutralbeams, and sensitivity is improved for highermasses.
The use of SIMS for moderate-size (3000-13,000Da) proteins and peptides has largely beensupplanted by electrospray ionization.
S d I M S t t (SIMS)
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Secondary Ion Mass Spectrometry (SIMS)
Sample introduction
Same as for FAB
Benefits
Same as for FAB, except sensitivity is improved forhigher masses (3000 to 13,000 Da).
S d I M S t t (SIMS)
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Secondary Ion Mass Spectrometry (SIMS)
Limitations
Same as for FAB except
target can get hotter than in FAB due to moreenergetic primary beam
high-voltage arcs more common than FAB
ion source usually requires more maintenance thanFAB
Mass range Moderate Typically 300 to 13,000 Da.
Electrospray Ionization (ESI)
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Electrospray Ionization (ESI)
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 tonebulize or aerosolize
Aerosol is directed through regions of highervacuum until droplets evaporate to near atomic
size (still carrying charges)
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Electrospray Ionization (ESI)
El t I i ti (ESI)
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Electrospray Ionization (ESI)
Can be modified to nanospray system
with flow < 1mL/min
Very sensitive technique, requires lessthan a picomole of material
Strongly affected by salts & detergents
Positive ion mode measures (M + H)+ (addformic 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 thatreadily accept H+ (such as amide andamino groups found in peptides andproteins) then positive ion detection isused-PROTEINS
If a sample has functional groups that
readily lose a proton (such as carboxylicacids and hydroxyls as found in nucleicacids and sugars) then negative iondetection is used-DNA
Electrospray Ionization (ESI)
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Electrospray Ionization (ESI)
Sample introduction
flow injection LC/MS
typical flow rates are less than 1 microliter perminute up to about a milliliter per minute
Electrospray Ionization (ESI)
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Electrospray Ionization (ESI)
Benefits
good for charged, polar or basic compounds permits the detection of high-mass compounds at
mass-to-charge ratios that are easily determined
by most mass spectrometers (m/z typically less
than 2000 to 3000). best method for analyzing multiply charged
compounds
very low chemical background leads to excellentdetection limits
can control presence or absence of fragmentation
by controlling the interface lens potentials
compatible with MS/MS methods
Electrospray Ionization (ESI)
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Electrospray Ionization (ESI)
Limitations
multiply charged species require interpretation andmathematical transformation (can sometimes bedifficult)
complementary to APCI. No good for uncharged,
non-basic, low-polarity compounds (e.g.steroids) very sensitive to contaminants such as alkali
metals or basic compounds
relatively low ion currents relatively complex hardware compared to other ion
sources
Mass range
Low-hi h T icall less than 200 000 Da.
Atmospheric Pressure Chemical Ionization
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Atmospheric Pressure Chemical Ionization
(APCI)
Similar interface to that used for ESI.
In APCI, a corona discharge is used to ionize theanalyte in the atmospheric pressure region.
The gas-phase ionization in APCI is more effective
than ESI for analyzing less-polar species.
ESI and APCI are complementary methods.
Atmospheric Pressure Chemical Ionization
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Atmospheric Pressure Chemical Ionization
(APCI)
Sample introduction
same as for electrospray ionization
Benefits
good for less-polar compounds
excellent LC/MS interface
compatible with MS/MS methods
Atmospheric Pressure Chemical Ionization
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Atmospheric Pressure Chemical Ionization
(APCI)
Limitations
complementary to ESI
Mass range
Low-moderate Typically less than 2000 Da.
Matrix-Assisted Laser Desorption
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Matrix-Assisted Laser Desorption
Ionization (MALDI)
The analyte is dissolved in a solution containing anexcess of a matrix such as sinapinic acid ordihydroxybenzoic acid that has a chromophorethat absorbs at the laser wavelength.
A small amount of this solution is placed on thelaser target.
The matrix absorbs the energy from the laserpulse and produces a plasma that results invaporization and ionization of the analyte.
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MALDI Ionization
++
+
+
-
--
++
+
+
-
---++
Analyte
Matrix
Laser
+
++
Absorption of UV radiationby chromophoric matrix andionization of matrix
Dissociation of matrix,phase change to super-compressed gas, chargetransfer to analyte molecule
Expansion of matrix atsupersonic velocity, analytetrapped in expanding matrixplume (explosion/popping)
+
+
+
MALDI
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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
P i i l f MALDI TOF MASS
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Principal for MALDI-TOF MASS
+
+
+++
+
+
+
+
+
pulsedUV or IR laser(3-4 ns)
detector
vacuum
strong
electricfield
Time Of Flight tube
peptide mixture
embedded inlight absorbingchemicals (matrix)
cloud ofprotonatedpeptide moleculesacc
V
P i i l f MALDI TOF MASS
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Principal for MALDI-TOF MASS
Linear Time Of Flight tube
Reflector Time Of Flight tube
detector
reflector
ion source
ion source
detector
time of flight
time of flight
Matrix-Assisted Laser Desorption
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Matrix Assisted Laser Desorption
Ionization (MALDI)
Sample introduction
direct insertion probe
continuous-flow introduction
Benefits
rapid and convenient molecular weightdetermination
Matrix-Assisted Laser Desorption
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Matrix Assisted Laser Desorption
Ionization (MALDI)
Limitations
MS/MS difficult
requires a mass analyzer that is compatible withpulsed ionization techniques
not easily compatible with LC/MS
Mass range
Very high Typically less than 500,000 Da.
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How is the sample introduced into the
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Mass Spectrometer?
For EI and CI there are four main options Heated reservoir septum inlet for pure gases or
volatile liquids. Basically this is a heated reservoir
(~200oC) with a small restriction bleed into the ion
source. Sample is injected into the reservoir
through a septum.
Direct insertion probe for volatile solids. Sample isloaded into a quartz tube at the end of a probe and
inserted directly into the ion source. The end of the
probe can then be heated, if required, up to
temperatures in excess 400oC.
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How is the sample introduced into the
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Mass Spectrometer?
For EI and CI there are four main options Particle Beam interface for semi-volatile compounds that
are amenable to EI and CI.
Samples are dissolved in a suitable solvent and the solution
is introduced into the mass spectrometer using a suitable(e.g. HPLC) pump.
The liquid is nebulised with helium gas to form an aerosol of
solvent droplets. The stream of liquid droplets passes
through a desolvation chamber and then a series of nozzles
and skimmers that remove the solvent and helium, allowing
a stream of solid particles (the sample) to enter the EI/CI ion
source.
How is the sample introduced into the
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Mass Spectrometer?
For FAB / LSIMS Sample can be dissolved directly into the matrix on thetarget or applied dissolved in a miscible solvent. The probeis then inserted in to the mass spectrometer. A dynamicversion of FAB exists, often referred to as continuous flowFAB. Here, the sample, in matrix, is introduced from outsidethe instrument and is then pumped to the target via a lengthof fused silica tubing. Interfacing chromatographictechniques such as HLPC and CZE has been successfully
achieved with this option, although practical difficulties areoften encountered. Dynamic FAB has largely beensuperseded by ESI, which is a more robust technique.
How is the sample introduced into the
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Mass Spectrometer?
FOR MALDI:The sample is dissolved in matrix, spotted onto the
target, allowed to crystallise and then inserted intothe instrument
How is the sample introduced into the
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Mass Spectrometer?
For ESI For pure samples dissolved in the mobile phase, a directinjection loop can be used. The technique really comes intoits own however, when interfaced to chromatographicprocedures such as CZE, CEC and in particular HPLC.Historically, mass spectrometry and HPLC have beenunhappy bedfellows. ESI has changed all that and the twoare now routinely and reliably interfaced together. Also, withsmaller columns and ever decreasing flow rates being used,
the amount of sample required for successful analyses isalso reduced, thus effectively increasing the sensitivity of thetechnique dramatically. With CEC being coupled to ananospray (e.g. 50nl/min) ESI interface, attomole detection
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