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1 Chemical Analysis by Chemical Analysis by Mass Spectrometry Mass Spectrometry Dr Phil Mortimer Dr Phil Mortimer Chemistry Department Mass Chemistry Department Mass Spectrometry Facility Spectrometry Facility 410-516-5552 410-516-5552 m m a a [email protected] [email protected]

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Page 1: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

11

Chemical Analysis by Chemical Analysis by Mass SpectrometryMass Spectrometry

Dr Phil MortimerDr Phil Mortimer

Chemistry Department Mass Chemistry Department Mass Spectrometry FacilitySpectrometry Facility

410-516-5552410-516-5552

[email protected]@jhu.edu

Page 2: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

22

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

Page 3: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

33

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.

Page 4: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

44

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

Page 5: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

55

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.

Page 6: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

66

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

Page 7: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

77

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

Page 8: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

88

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?

Page 9: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

99

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

Page 10: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1010

Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

Page 11: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1111

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

Page 12: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1212

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

Page 13: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1313

Resolution

Influences achievable precision and accuracy of measurement

Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

Page 14: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1414

Resolution

Influences achievable precision and accuracy of measurement

R = ΔM/M

Often expressed in ppm

R = (ΔM/M) x106

Page 15: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1515

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

Page 16: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1616

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.

Page 17: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1717

What is a Mass Spectrometer?

All Instruments Have:

1. Sample Inlet

2. Ion Source

3. Mass Analyzer

• Detector

• Data System

Page 18: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1818

e-

+

e-

e-

+4000 V 0 V

+

e-

e-

heavy

light

Magnetic and/or electric field

sample

vapourise

ioniseaccelerate separate

+A

+B

+C

A+ B+ C+

vacuum

Mass spectrometryMass spectrometry

How does it work?

Page 19: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

1919

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

Page 20: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2020

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

Page 21: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2121

Page 22: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2222

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

Page 23: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2323

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

Page 24: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2424

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.

Page 25: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2525

Analyser Types – Quadrupole ion Trap

Ion Trap is very small – most of instrument is ion guides into the trap itself

Bruker HCT

Page 26: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2626

Analyser Types – Time of Flight (ToF)

Conceptual diagram!!!

Page 27: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2727

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 detector for constant E

Page 28: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2828

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

Page 29: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

2929

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

Page 30: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3030

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

Page 31: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3131

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).

Page 32: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3232

Electron Impact (EI) Mass Spectrometry

Page 33: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3333

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.

Page 34: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3434

CI-MS

Comparison of EI and CI spectra

Page 35: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3535

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.

Page 36: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3636

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)

Page 37: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3737

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.

Page 38: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3838

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 (can be 20nM-50uM depending on sample)

Usually requires addition of volatile buffer (0.1-1%)

Typically AcOH or TFA (+ve ion) / NH4OH (-ve ion)

Page 39: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

3939

Electrospray Mass Spectrometry (ESI-MS)

How does it work?

Page 40: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4040

Electrospray Mass Spectrometry (ESI-MS)

How does it work?

Page 41: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4141

Electrospray Mass Spectrometry (ESI-MS)

Thermo-Finnigan LCQ-Deca

ESI-Ion Trap with LC System

Page 42: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4242

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

Page 43: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4343

Electrospray Mass Spectrometry (ESI-MS)

Can Deconvolute mass spectra as previously discussed

Page 44: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4444

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

Page 45: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4545

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

Page 46: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4646

MALDI-ToF Mass Spectrometry

Page 47: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4747

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

Page 48: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4848

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

Page 49: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

4949

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 prepMultiple layer / co-mixed

Spot 0.5uL of mixture on spot and allow to dry

Analysis very dependant upon sample preparation

Page 50: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5050

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

MALDI-ToF Mass Spectrometry - Matrices

Page 51: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5151

MALDI Contamination LimitsAnalysis is relatively insensitive to contaminants.

Phosphate 20 mM EDTA 1 mMDetergents 0.1% Glycine 20 mMGlycerol 2% Sodium Citrate 20 mMBuffer (Tris)50 mM K phosphate 25 mMGuanidine 1 M Na phosphate 0.1MNa azide 1% Octyl glucoside 0.3%SDS 0.05% Ammon. Bicarb. 0.1M

Suggested concentrations ~10 pmol @ <10 000 Da (pure) ~100 pmol @ >50 000 Da (pure)

Page 52: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5252

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

Page 53: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5353

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

Page 54: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5454

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 determinationProtein Identification and profiling using digests and data base

searching – major development in ProteomicsProtein post-translational modification Protein structure characterisation

Maldi-Imaging

Oligo-nucleotide analysis – Confirmation of purity of synthetic oligo’s

Carbohydrate analysis

Page 55: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5555

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)

Page 56: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5656

Mouse Brain Digital Photo Before Matrix Addition

Page 57: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5757

Mouse Brain H&E Stain After Molecular Imaging

Page 58: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5858

m/z5000 10000 15000 20000 25000

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

a.u.

600-30,000 Da

Mouse Brain Full Molecular Spectrum

Page 59: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

5959

Molecular Image of Lipid Mass m/z = 786

Page 60: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

6060

Molecular Image of Lipid Mass m/z = 1493

Page 61: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

6161

Molecular Image after Unsupervised PCA

Page 62: 1 Chemical Analysis by Mass Spectrometry Dr Phil Mortimer Chemistry Department Mass Spectrometry Facility 410-516-5552 mass.spec@jhu.edu ass.spec@jhu.eduass.spec@jhu.edu

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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 protocolsPay 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?