1. Chemical Analysis by Mass Spectrometry
Dr Phil Mortimer
Chemistry Department Mass Spectrometry Facility

2. Recommended Reading :
“The Expanding Role of mass Spectrometry in Biotechnology”
Gary Siuzdak, MCC Press, San Diego, ISBN
“Ionization Methods in Organic Mass Spectrometry”
Alison Ashcroft, RSC, Cambridge, UK, ISBN
“Practical Organic Mass Spectrometry” 2nd Edn
J R Chapman, Wiley, Chichester, UK, ISBN X
“Spectroscopic Methods in Organic Chemistry” 4th Edn
D H Williams, I Fleming, McGraw-Hill, ISBN X

3. 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.

4.  Can work out molecular structure
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

5. 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.

6. 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, 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

7. 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

8. 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?

9. 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 = ( )/1 = 10001
When z = 2 m/z = ( )/2 =
When z = 3 m/z = ( )/3 =
When z = 4 m/z = ( )/4 =
When z = 5 m/z = ( )/5 =

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

11. 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

12. 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 =
(weighted average for each isotope)
Exact mass =
(exact mass of most abundant isotope)
Nominal mass = 1442
(integer mass of most abundant isotope)
Illustrated on next Slide

13. Resolution Influences achievable precision and accuracy of measurement
Figure from The Expanding Role of MS in Bio-technology – G . Siuzdak

14. Resolution Influences achievable precision and accuracy of measurement
R = ΔM/M
Often expressed in ppm
R = (ΔM/M) x106

15. 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

16. 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.

17. What is a Mass Spectrometer?
All Instruments Have:
Sample Inlet
Ion Source
Mass Analyzer
Detector
Data System

18. How does it work? +4000 V 0 V e- e- e- e- e- Mass spectrometry
accelerate
separate
ionise
+4000 V
0 V e-
Magnetic and/or electric field e- + +
heavy
vacuum
light
vapourise e- + A e-
sample + B + C A+ B+ C+
Mass spectrometry e-

19. 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

20. 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

22. 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

23. 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

24. 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.

25. Analyser Types – Quadrupole ion Trap
Bruker HCT
Ion Trap is very small – most of instrument is ion guides into the trap itself

26. Analyser Types – Time of Flight (ToF)
Conceptual diagram!!!

27. 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

28. 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

29. 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

30. 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

31. Electron Impact (EI) Mass Spectrometry
Problems with EI ionisation
– 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).

32. Electron Impact (EI) Mass Spectrometry

33. Overcoming problems with EI-MS – Use of CI
How to overcome limitations?
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.

34. CI-MS
Comparison of EI and CI spectra

35. 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.

36. 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)

37. 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.

38. 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)

39. Electrospray Mass Spectrometry (ESI-MS)
How does it work?

40. Electrospray Mass Spectrometry (ESI-MS)
How does it work?

41. Electrospray Mass Spectrometry (ESI-MS)
Thermo-Finnigan LCQ-Deca
ESI-Ion Trap with LC System

42. 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

43. Electrospray Mass Spectrometry (ESI-MS)
Can Deconvolute mass spectra as previously discussed

44. 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

45. 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

46. MALDI-ToF Mass Spectrometry

47. 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

48. 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

49. MALDI-ToF Mass Spectrometry - Conditions
Suggested concentrations
~10 < Da (pure)
~100 > 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

50. 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

51. MALDI Contamination Limits
Analysis is relatively insensitive to contaminants.
Phosphate 20 mM EDTA 1 mM
Detergents 0.1% Glycine 20 mM
Glycerol 2% Sodium Citrate 20 mM
Buffer (Tris)50 mM K phosphate 25 mM
Guanidine 1 M Na phosphate 0.1M
Na azide 1% Octyl glucoside 0.3%
SDS 0.05% Ammon. Bicarb. 0.1M
Suggested concentrations
~10 < Da (pure)
~100 > Da (pure)

52. 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

53. 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

54. 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

55. 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)

56. Mouse Brain Digital Photo Before Matrix Addition

57. Mouse Brain H&E Stain After Molecular Imaging

58. Mouse Brain Full Molecular Spectrum
600-30,000 Da

59. Molecular Image of Lipid Mass m/z = 786

60. Molecular Image of Lipid Mass m/z = 1493

61. Molecular Image after Unsupervised PCA

62. 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.

63. Any Questions?