1. Mass Spectrometry Workshop

2. Processes in MS MS involves three distinct processes:
Volatilisation of the sample
Ionisation of the molecules
Separation and detection of the ions according to m/z ratio
Mass
separation
Ionisation
Sample
inlet
Source
Mass
analyser
Detector
Data
System
Vacuum System

3. Ionisation
Both positive and negative ions can be formed by MS techniques, but positive ions are most commonly used. The ions usually have one positive charge.
There are several methods of ionisation, but the most common these days are electrospray (ESI, small/large molecules) and matrix-assisted laser desorption (MALDI, large molecules)

4. Electrospray (ESI)
This is the most popular technique for ionising samples soluble in polar solvents (H2O, MeOH and EtOH). It is therefore very important for the analysis of biomolecules of high molecular weight. A derivative of this technique, atmospheric pressure chemical ionisation (APCI) can be used to ionise samples soluble in organic solvents (CH2Cl2, hexanes etc).
The main problems arise from the existence of multiply charged ions (which is an advantage for large molecules!), and the presence of other quasi-molecular ions:
[M + MeOH + H]+, [M + Na]+, [M + K]+

5. Electrospray (ESI)
The dissolved sample is pumped through a steel capillary at a rate of 1-20 mL/min which is at +3 kV. The electric field at the tip of the capillary charges the surface of the emerging droplets. The solvent is evaporated using N2, reducing the size of the droplet, until the charges come into close proximity, and a Coulombic explosion occurs giving rise to a quasi-molecular ion of the form [M + H]n+ where n can be >1.

6. Electrospray (ESI)

7. Matrix Assisted Laser Desorption (MALDI)
Enables gentle ionisation of very large molecules with a single charge
Compound dissolved in matrix and deposited in ‘target’
Irradiated by laser beam – energy absorbed by matrix and transferred to analyte which is desorbed.

8. Mass Analysis There are many types of mass analyser:
Magnetic sector/electric sector
Quadrupole
Ion Trap
Time-of-flight
Fourier Transform Ion Cyclotron Resonance (FT-MS)
Orbitrap

9. Mass analyser: Orbitrap – an electrostatic trapz φ r
ions trapped in an electrostatic field
central electrode kept on high voltage
outer electrode is split and able to pick up an image current induced by ion packets moving inside the trap
source:

10. Principle of ion trap ESI-LC-MS
LTQ Orbitrap Discovery: hybrid system combining LTQ XL linear ion trap MS and Orbitrap mass analyzer
ions generated in the API ion source are trapped in the LTQ XL, and analyzed using the MS and MSn scan modes
source:

11. Principle of ion trap ESI-LC-MS
ions are axially ejected from the LTQ XL and collected in a C-shaped ion trap (C-Trap) from which they are passed into the Orbitrap mass analyzer
ions transferred from the C-Trap are captured by rapidly increasing the voltage on the center electrode of the Orbitrap
trapped ions assume circular trajectories around the center electrode and their axial oscillations, along the center electrode, are detected
source:

12. Mass analyser: time-of-flight (TOF)
ions are accelerated at different velocities depending on their m/z ratios
ions of lower masses are accelerated to higher velocities and reach the detector first
the m/z ratio for each ion is calculated based on its time-of-flight, t
source:

13. Monoisotopic masses
Isotope
Mass
% Abundance 1H
1.008
99.99
12C
12.000
98.89
13C
13.003
1.11
14N
14.003
99.64
15N
15.000
0.36
16O
15.995
99.76
18O
17.999
0.20
32S
31.972
95.03
33S
32.971
0.76
34S
33.968
4.20
35Cl
34.969
75.77
37Cl
36.966
24.23
79Br
78.918
50.52
81Br
80.916
49.48
For many elements, there is more than one isotope, each with a different natural abundance. For the most common elements in organic molecules:

14. Isotope Ratio Patterns
For hydrocarbons, the height of the [M + 1] peak is given by the expression:
[M + 1] = n x 1.1%
where n is the number of carbon atoms. The height of the [M + 1] peak can therefore be used as a crude measure of the number of carbon atoms in a hydrocarbon.
For most other compounds, [M + 2] is very small, apart from those containing Cl or Br. The region around the molecular ion peak becomes more complex if either of these (or both) are present.

15. Cl and Br 2 Br atoms in the same molecule: (1 79Br + 1 81Br)2 =
1 79Br79Br Br81Br Br81Br
2 Cl atoms present:
(3 35Cl Cl)2 =
9 35Cl35Cl Cl37Cl Cl37Cl

16. Fragmentation

17. Allowed Fragmentations
Thermodynamics dictates that even-electron MS ions cannot cleave to a pair of odd-electron fragments
Even = even-electron ions in which all outer shell electrons are paired. EIMS give odd-electron molecular ions, [M]+. and ionization methods such ESI or CI give even-electron molecular ions, [M+H]+

18. Multiply Charged Ions

19. Peptides and Proteins

20. Multiply Charged Ions [M + H]+ [M + 2H]2+ [M + 3H]3+ [M + nH]n+
Using electrospray ionization it is common to get multiply charged ions:
[M + H]+ [M + 2H]2+ [M + 3H]3+ [M + nH]n+
m/z: M (M+2)/2 (M+3)/3 (M+n)/n

21. Isotope Ratio Patterns: C100H200
For 12Cm13Cn m n
1403.6
1404.6 2
Chemputer

22. MALDI-TOF Data

23. Determining the Molecular Formula

24. Accurate Mass Measurement
Experimental accurate mass measurement (from MS) was suggesting C10H16 is the correct formula.
The error between calculated and experimental mass is:
= = 0.8 mmu
Formula
dbe
Accurate mass
C10H16 3
C9H12O 4
C8H8O2 5
C7H4O3 6
C9H14N
3.5
C8H12N2

25. MS Errors
Experimental accurate mass measurement (from MS) was suggesting C10H16 is the correct formula.
The error between calculated and experimental mass is:
= = 0.8 mmu
Formula
dbe
Accurate mass
C10H16 3
C9H12O 4
C8H8O2 5
C7H4O3 6
C9H14N
3.5
C8H12N2

26. Resolution R
R = m/Dm
Defined by the width of a peak at a specific fraction of the peak height.
Peaks are separated if the valley goes down to 10%
Some manufacturers define 50%

27. Resolving Power

29. LC-MS

30. LC-MS Profiling Modestobacter
marine biodiscovery centre
Unknown

31. MS-MS

33. Fragments Confirm Structure
marine biodiscovery centre
Double bond stereochemistry?
Stereocentres at C10 and C11

34. Other MS-MS modes

35. Peptide Fragmentation

38. Unknown compound
marine biodiscovery centre
MS-MS
Pro
Leu/Ile
Glu

39. Use of MS-MS Data

40. Streptomyces CT34
marine biodiscovery centre

41. LC-MS of CT-34
marine biodiscovery centre
MW = 3600

42. Peptidogenomics
Dorrestein et al Nat Chem Biol. 2012, 7, 794–802

43. Sequence tags
marine biodiscovery centre
Dorrestein lab, UCSD

44. Gene Cluster

45. Molecular Formula
marine biodiscovery centre

46. Overlapping MS Fragments
z-ions
x-ion
y-ions
Seq Tags
Me2N-I-X-P-L-A-X-L-A-X-P-E-A-X-P-V-G-F-A-A-X-S-A-X-A-A-A-V-N-M-I-X-H-D-V-X-R-H-OH
b-ions
a-ions
c-ion

47. Using Chemistry to Guide Strain Selection
marine biodiscovery centre

48. MALDI Biotyping
marine biodiscovery centre

49. PCA analysis/clustering
marine biodiscovery centre

50. Xcalibur Spectrum window
Layouts (provide software and standard layouts)
Verifying a peak is a peak
Setting ranges
Checking negative mode data
Doubly charged ions (Ada 1113)

51. Xcalibur Accurate mass determination Fragments
Setting limits (Vuaa example - 338)
Using sodiated ions (same example)
Isotope patterns (Se example)
Fragments
Using the MSn data (use acl8 844 example)
Setting the [M+H]+ to zero
Relative mass differences vs MF of fragments

52. Exercise – Linear Peptides

53. Exercise – Cyclic Peptides

54. Exercise – for each peptide:
Find peak corresponding to molecular ion
Confirm molecular formula and error (ppm)
Use accurate mass MSn fragments to confirm sequence of amino acids.
For linear peptides explain fragments using b and y ions.
For cyclic peptides explain possible fragments
For patellamide D explain unusual fragments.