Transcript Mass Spectrometry in BioTechnology

Chemical Analysis by
Mass Spectrometry
Dr Phil Mortimer
Chemistry Department Mass
Spectrometry Facility
410-516-5552
[email protected]
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Recommended Reading :
“The Expanding Role of mass Spectrometry in Biotechnology”
Gary Siuzdak, MCC Press, San Diego, ISBN 0-9742451-0-0
“Ionization Methods in Organic Mass Spectrometry”
Alison Ashcroft, RSC, Cambridge, UK, ISBN 0-85404-570-8
“Practical Organic Mass Spectrometry” 2nd Edn
J R Chapman, Wiley, Chichester, UK, ISBN 0-471-95831-X
“Spectroscopic Methods in Organic Chemistry” 4th Edn
D H Williams, I Fleming, McGraw-Hill, ISBN 0-07-707212-X 2
Chemistry 101
• All chemical substances are combinations of atoms.
• Atoms of different elements have different masses (H = 1, C =
12, O = 16, S = 32, etc.)
• An element is a substance that cannot be broken down into a
simpler species by chemical means - has a unique atomic
number corresponding to the number of protons in the nucleus
• Different atoms combine in different ways to form molecular
sub-units called functional groups.
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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-to-charge 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
• But this is not always true
For large bio-molecules analysed by electrospray (ESI), z >1
What happens in this case?
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Multiple Charging
Consider a peptide with MW of 10000
With ESI-MS, charges by H+ addition
M + nH+  MnHn+
Resultant ions formed are :When z = 1
m/z = (10000+1)/1 = 10001
When z = 2
m/z = (10000+2)/2 =
5002
When z = 3
m/z = (10000+3)/3 =
3334.3
When z = 4
m/z = (10000+4)/4 =
2501
When z = 5
m/z = (10000+5)/5 =
2001
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Figure from The Expanding Role of MS
in Bio-technology – G . Siuzdak
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Multiple Charging
Advantage in that allows measurement of high mass ions
with instruments of limited m/z range.
Particularly true for ESI-MS – Advantage for analysis of high
mass samples that take multiple charges – brings sample
m/z down into measurable range of MS
Computer Algorithms deconvolute m/z to original mass.
Figure from The
Expanding Role of MS
in Biotechnology – G .
Siuzdak
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Mass Measurement
Mass Spectrometers measure isotopic mass.
They DO NOT measure average molecular mass!! (MW)
e.g For a molecule with empirical formula C60H122N20O16S2
Average MW = 1443.8857
(weighted average for each isotope)
Exact mass
= 1442.8788
(exact mass of most abundant isotope)
Nominal mass = 1442
(integer mass of most abundant isotope)
Illustrated on next Slide
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Resolution
Figure from The Expanding Role of MS
in Bio-technology – G . Siuzdak
Influences achievable precision and accuracy of measurement
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Resolution
R = ΔM/M
Often expressed in
ppm
R = (ΔM/M) x106
Influences achievable precision and accuracy of measurement
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Isotope Patterns
Isotope patterns
useful for
identifying presence
of certain elements
Particularly useful
for SMALL
molecules
Figure from The Expanding Role of MS
in Bio-technology – G . Siuzdak 15
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
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and development of modern powerful analytical
techniques.
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
+4000 V
e-
separate
0V
Magnetic and/or
electric field
e+
vapourise
e-
heavy
+
v
a
c
u
u
m
light
A+
esample
+
B
+
Mass spectrometry
C
e-
A+
B+
C+
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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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Analyser Types – Quadrupole
Smaller, cheaper – computer controlled – Self service
operation by trained users possible
Electrostatic momentum separation by superimposed rf and
dc voltages
Rapid scan rates – enables measurement of transient
samples introduced from chromatographic systems (GC, LC)
Lower resolution – accurate mass NOT possible
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Analyser Types – Quadrupole ion Trap
Derivative of Quadrupole – cheap, small, rapid scanning
Again, electrostatic momentum separation by rf and dc
voltages
Lower resolution – accurate mass not possible
BUT – have ion trapping ability – can store and selectively
eject ions
Ions can be subjected to fragmented by CID and “daughter
ions” analysed
Allows MS-MS or MSn (Multiple levels of storage and
trapping)
Can perform both molecular ion analysis and structural
determination
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Analyser Types – Quadrupole ion Trap
3 Electrode system
2 x Endcap and 1x Ring Electrode
Now have recent develpoment of
Linear Ion Trap and orbitrap
Developments on same theme.24
Analyser Types – Quadrupole ion Trap
Bruker HCT
Ion Trap is very small – most of instrument is ion
guides into the trap itself
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Analyser Types – Time of Flight (ToF)
Conceptual diagram!!!
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Analyser Types – Time of Flight (ToF)
Velocity separation - E= mv2
Ion packet given constant KE – ions of heavier mass take
longer to pass down drift tube and reach detector
Conceptually easy
Allows very large masses to be measured (500,000Da)
E= 1/2mv2
Time flight of ions
through drift tube
Ions of larger mass
take longer to reach
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detector for constant
E
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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Development of Mass Spectrometry
Until 1980’s, most mass spec geared primarily towards
“traditional” chemical analysis (small molecules)
- MS primarily conducted using EI ionisation – unchanged
since 30’s and 40’s
From 1980’s, start to have shift in focus towards analysis of
samples that are larger and more bio-molecular in character
Such samples are often more delicate and easily fragmented.
This results in the development of “softer” ionisation techniques
and analysers capable of extended mass ranges.
Allows MS determination of high mass parent ions (such as intact
proteins, etc.).
Strongly influences development of Proteomics field
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Electron Impact (EI) Mass Spectrometry
Up until 1980’s, most mass spec is “chemical” analysis - performed
using EI ionisation
Bombard gaseous sample with high energy (70eV) e-
Results in ejection of e- from target molecule to form gas phase ion
species – which is then passed to analyser for analysis.
e- + M -> 2e- +M+
Sample normally introduced via heated probe, GC, or leak (frit) inlet
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Electron Impact (EI) Mass Spectrometry
Problems with EI ionisation
1) – requires sample be in the gas phase before ionisation
- limits samples to those already existing in the gas phase or
thermally stable samples that are easily volatised (for probe
introduction)
2) – High Energy (Hard) Ionisation – lots of excess energy given
to target – causes fragmentation to lose energy and become
stable – resulting in lots of characteristic fragments ions, but
little parent ion (useful for structural characterisation).
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Electron Impact (EI) Mass Spectrometry
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Overcoming problems with EI-MS – Use of CI
How to overcome limitations?
1) Derivatize sample to make more volatile and thermally stable
derivative that can be analysed by EI
2) Develop other ionisation techniques using lower ionisation
energies and other means of introducing sample.
Intermediate method was Chemical Ionisation (CI)
Uses bath gas (CH3/NH4/CH3(CH2)2CH3) to protonate sample
Often forms MH+
Still only applicable to volatile or Thermally stable samples.
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CI-MS
Comparison of EI and CI spectra
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FAB-Mass Spectrometry
Subsequent development of FAB (Fast Atom Bombardment)
Still used for small delicate molecules
Dissolve sample in liquid matrix and place on target
Bombard with beam of fast atoms or ions (Xe or Cs+)
Have secondary ion emission
Low energy protonation of target molecules – very little excess
energy – little fragmentation – readily observe parent ions.
Now we’re getting somewhere.
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FAB-Mass Spectrometry
Problems with FAB
Slow, Labor intensive, Very
skilled.
Matrix interference at low
mass
Generally observe
MH+ (+ve ion mode)
OR
M-H (-ve ion mode)
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Current Mass Spectrometry – Biochemical MS
Today, majority of MS is of bio-chemiccal / biological samples
performed using either Electrospray MS or Maldi-toF MS.
Other methods exist, but these perform bulk of the work
Will concentrate on these for the rest of the lecture.
Both are “soft” (low energy) ionisation methods that usually yield
little fragmentation and so are useful for determination of
parent mass of delicate molecules.
Both are condensed phase techniques and require that samples
are soluble.
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Electrospray Mass Spectrometry (ESI-MS)
Solution phase technique - Can analyse both +ve and –ve ions
(but not simultaneously)
Samples usually dissolved in moderately polar solvent
Typically MeOH or MeCN, often mixed H2O (up to 80%)
DO NOT USE DMF, DMSO, THF, etc
Do NOT use involatile buffers.
Typical concentration 1-10uM
sample)
(can be 20nM-50uM depending on
Usually requires addition of volatile buffer (0.1-1%)
Typically AcOH or TFA (+ve ion) / NH4OH (-ve ion)
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Electrospray Mass Spectrometry (ESI-MS)
How does it work?
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Electrospray Mass Spectrometry (ESI-MS)
How does it work?
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Electrospray Mass Spectrometry (ESI-MS)
Thermo-Finnigan LCQ-Deca
ESI-Ion Trap with LC System
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Electrospray Mass Spectrometry (ESI-MS)
Different versions of ESI (On-Axis / Orthoganal / Off Axis)
Advantages
Soft ionisation – limited fragmentation
Multiple charging with peptides / proteins / oligionucleotides
(Analysis of molecules with MW > mass range of
instrument)
Can be linked with LC – acts as inlet – allows MS
identification of components of mixtures
Automated high throughput analysis of biological samples –
24/7
Can be coupled with many analysers – IT/Quadrupole /ICR /
Orbitrap – vast range of different types of analysis possible
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Electrospray Mass Spectrometry (ESI-MS)
Can Deconvolute mass spectra as previously discussed
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MALDI-ToF Mass Spectrometry
Relatively simple technique
Soft ionisation method that can be used to volatilise large
macromolecules with minimum fragmentation
Gives less multiple charging than ESI
Samples co-deposited on target plate with matrix (and often
an additive) and allowed to dry.
Many samples can be on plate.
Plate inserted into instrument vacuum
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MALDI-ToF Mass Spectrometry
Target irradiated by UV laser.
Causes vaporisation of matrix and supersonic expansion of
plume
Dried sample is launched into the gas phase as matrix is
vaporised
UV energy absorbed by matrix causes it to dissociate and
typically transfers a proton to sample molecule within the
plume to form MH+
Now have protonated target, which is accelerated into analyser
for seperation and detection
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MALDI-ToF Mass Spectrometry
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MALDI-ToF Mass Spectrometry
Most MALDI-ToF are
reflectron instruments
Reflectron is energy
focusing device (ion mirror)
Increases resolution (and
mass accuracy) – but limits
mass range
Linear ToF has low
resolution but high mass
range (up to m/z 300,000)
Many Instruments are now
ToF/ToF
Can do MS/MS experiments
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MALDI-ToF Mass Spectrometry
Typical Current State of the
Art Maldi-ToF
Bruker Autoflex
Now available as Tof/ToF
Easy to use – walk up use
after training.
Highly automated
Now can be used for
imaging of Tissue samples
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MALDI-ToF Mass Spectrometry - Conditions
Suggested concentrations
~10 pmol @ <10 000 Da (pure)
~100 pmol @ >50 000 Da (pure)
10: 1 Ratio of Matrix : Sample
(20nM-50uM of sample – typically 1-10uM)
Several methods of target prep
Multiple layer / co-mixed
Spot 0.5uL of mixture on spot and allow to dry
Analysis very dependant upon sample preparation
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MALDI-ToF Mass Spectrometry - Matrices
Matrix
Application
α-Cyano-4-hydroxycinnamic acid
(CCA)
peptides
3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic
acid)
proteins
2,5 Dihydroxybenzoic acid (DHB)
peptides,
proteins,
polymers, sugars
3-Hydroxypicolinic acid (HPA)
oligonucleotides
Dithranol (anthralin)
polymers
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MALDI Contamination Limits
Analysis is relatively insensitive to contaminants.
Phosphate 20 mM
Detergents 0.1%
Glycerol
2%
Buffer (Tris)50 mM
Guanidine 1 M
Na azide
1%
SDS
0.05%
EDTA
Glycine
Sodium Citrate
K phosphate
Na phosphate
Octyl glucoside
Ammon. Bicarb.
1 mM
20 mM
20 mM
25 mM
0.1M
0.3%
0.1M
Suggested concentrations
~10 pmol @ <10 000 Da (pure)
~100 pmol @ >50 000 Da (pure)
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MALDI –Characteristics
Maldi-ToF Generally results in broader peak envelope than
ESI
This is particularly true at high mass.
Low mass Maldi-ToF (<20,000Da) – can use reflectron –
get high resolution (R>10,000)
High MW Maldi – requires use of linear mode – lower
resolution – Higher Mass range (up to 500,000Da
Maldi-ToF generally results in generation of singly charged
species (z = 1)
However, often requires desalting, otherwise have broad
mass envelop addition due to multiple slated peaks forming
– particularly prevalent for proteins
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MALDI –Characteristics
Analysis is rapid – therefore, is often used for high
throughput analysis and screening applications – many
samples on one plate.
Sensitivity enhanced by using “AnchorChip” Plates –
concentrates sample solution in small spot
Low mass spectra (<500MW) can be inhibited by
interference from Matrix peaks – development of Naldi
Spectra VERY dependant upon sample preparation and
analysis conditions (especially laser power) – modern
instruments have “fuzzy” logic to optimise analytical
conditions on the fly
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Biotechnology applications
Advances in Proteomics and other areas in biotechnology made
possible by development of soft ionisation Maldi and ESI MS
techniques
Protein and peptide analysis for MW determination
Protein Identification and profiling using digests and data base
searching – major development in Proteomics
Protein post-translational modification
Protein structure characterisation
Maldi-Imaging
Oligo-nucleotide analysis – Confirmation of purity of synthetic oligo’s
Carbohydrate analysis
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Biotechnology applications
Automated high throughput analysis
Screening of biological samples
Pharmicokinetics
LC-MS – seperation and identification of components of complex
mixtures – Normally LC-ESI, now increasingly LC-Maldi-ToF
Intact virus analysis
Cell imaging (Maldi)
Tissue Imaging (Maldi)
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Mouse Brain Digital Photo Before Matrix Addition
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Mouse Brain H&E Stain After Molecular Imaging
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Mouse Brain Full Molecular Spectrum
a.u.
6.5
600-30,000 Da
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5000
10000
15000
20000
25000
m/z
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Molecular Image of Lipid Mass m/z = 786
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Molecular Image of Lipid Mass m/z = 1493
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Molecular Image after Unsupervised PCA
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Practical Analytical MS Considerations
Know what you are trying to achieve –
Structural analysis? Accurate Mass Determination?
Prepare sample according to given preparation protocols
Pay attention to sample amount / concentration
Best results with purified samples – Mixtures of components
give reduced spectra intensity and difficult to identify sample
components
Remember : - you know most about your sample – not the
analyst – give any and all available required information.
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Any Questions?
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