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1. Lecture 5
Condensed phase ionisation
techniques: spray methods

2. At the end of this lecture you
should be able to:
•describe the ion formation models in
ESI-MS
•calculate molecular weights and charge
states from low-and high-resolution
ESI-MS spectra

3. Ionisation Techniques: Overview
Gas-Phase Methods
•Electron Impact (EI)
•Chemical Ionization (CI)
Desorption Methods
•Secondary Ion MS (SIMS) and Liquid SIMS
•Fast Atom Bombardment (FAB)
•Laser Desorption/Ionization (LDI)
•Matrix-Assisted Laser Desorption/Ionization (MALDI)
Spray Methods
•Atmospheric Pressure Chemical Ionization (APCI)
•Electrospray (ESI)

4. Condensed phase ionisation
techniques (2): spray methods
Overview
•Thermospray
•APCI: Atmospheric pressure chemical
ionisation
•APPI: Atmospheric pressure
photoionisation
•Electrospray ionisation

5. Atmospheric pressure chemical
ionisation (APCI)
•For non-polar and thermally stable compounds
< 1500 Da
•Useful to combine with liquid chromatography
www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
Flow
Spray needle/
Capillary Spray
Cone
Atmospheric pressure
Skimmers
Vacuum
To mass
spectrometer
Nebuliser gas
Corona discharge
Ions

6. Electrospray Ionisation (ESI)
•Nobel Prize to Fenn in 2002
•Also atmospheric pressure ionisation
•Very versatile
•Also works for (very) large (bio)molecules, including
proteins, nucleic acids, carbohydrates
•Softest ionisation technique of all
3-4 keV
2-10 ml/min
http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
Flow
Spray needle/CapillarySpray
Cone
Atmospheric pressure
Skimmers
Vacuum
To mass
spectrometer

7. Electrospray Ionisation (ESI)
•Flowrates: 2 to 10 ml/min: Best interface for
LC/MS
•Can be combined with almost any mass
analyser
Common: TOF, Ion Trap, Quadrupole, FT-ICR
•Uses: Mass detection, structure elucidation,
protein folding, H/D exchange, protein
sequencing….

8. Sample characteristics
•Common solvents: mixtures of water with
acetonitrile or methanol
•Usually with added acid (acetic, formic), < 1%
•Can’t tolerate (non-volatile) salt or buffers
•Can do positive or negative electrospray:
selected by capillary voltage

9. What it looks like

10. Ion evaporation model
Ionisation models
Charged residue model
www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
Analyte
molecule
Multiply charged
droplet
Spray
needle tip
Rayleigh limit
is reached
Multiply charged
droplet
1.Spray generates multiply
charged droplets
2.Solvent evaporation leads to
increasing charge density
3.When charge density too
high: Coulombic explosion

11. Charged amino acids
N
O
O
O
-
Aspartate
N
O
O
O
-
Glutamate
Acidic
3.65
4.24
N
O
N
NH
Histidine
6.00
N
O
N
N
N
+
ArginineN
OH
3N
+
Lysine
Basic
10.8
12.5
Usually negatively charged at pH 7
Positively/uncharged at pH 7
Always positively charged

12. Charges on surface
of proteins
Red: Negatively charged
Blue: Positively charged
White: No charge;
hydrophobic

13. Examples
•ESI of large molecules usually produces multiply
charged ions
•e.g. proteins:
•Each peak corresponds to the same protein, but
with different number of protons attached:
Observed ions are[M+nH]
n+
750 1000 1250 1500 1750 2000 2250 2500 2750
13+
12+
11+
10+
9+
8+
7+
6+
Charge state series
m/z

14. 942.8
893.3
848.7
808.3
771.6
738.1
707.4
998.1
1060.5
How to determine the molecular mass of a protein from
an ESI-MS spectrum
•Observed ions have composition
[M+nH]
n+
•Let m
1,m
2,…, m
n: m/z values of the
different peaks
•m
n=
•Its neighbouring peak to the left:
•m
n+1=
•Solving both equations for n and M:
•n =
•M = n(m
n–H)
•e.g. m
2= 998.1 and m
1= 1060.5
•n = 16, and M = 16952 Da
m/z
1n
1)H](n[M
+
++
n must be an integern
nH][M+ 1nn
1n
mm
Hm
+
+



15. Deconvolution of ESI mass spectra
Charge states
Deconvoluted
spectrum
M = n(m
n–H)
3000 30000
Mass (Da)

16. Quick reminder: average and
monoisotopic mass

17. The distance between isotopic
peaks reveals charge stateprotein_modelingLCTmix of 6 proteins
m/z
915 916 917 918
%
0
100
prot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374) TOF MS ES+
1.86e3915.7363
915.4818
915.2274
915.9765
916.2311
916.4857
916.7402 protein_modelingLCTmix of 6 proteins
m/z
1084 1085 1086 1087 1088 1089 1090
%
0
100
prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675) TOF MS ES+
4541086.5515
1086.0433
1087.0444
1087.5529
1088.0460 protein_modelingLCTmix of 6 proteins
m/z
500 501 502 503 504 505 506 507 508 509 510 511 512
%
0
100
prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651) TOF MS ES+
783505.3506
506.3584
507.3566
915.2247
915.4818
915.7363
915.9765
916.2311
916.4857
505.3506
506.3584
507.3566
1086.0433
1086.5515
1086.0444
1087.5529
1088.0460
+1
1.00
0.51
+2
0.25
+4
Jonathan A. Karty

18. 1695.7 1696.2 1696.7 m/z
0.0
0.5
1.0
1.5
a.i.
Charge States and distance between
isotopic peaks
0.1 +10

19. Example beyond molecular mass:
Protein folding
•Calbindin: Calcium binding induces protein folding: increase in
charge states with higher m/z (=lower charge, more folded)
No Ca
2+
excess Ca
2+
deconvolutedraw data

20. Recent developments:
ambient mass spectrometry:
DESI and DART
•DESI: Desorption electrospray ionisation
•DART: Direct analysis in real time
•Applicable to solids, liquids, and gases
•No prior sample treatment !

21. Ionsiation techniques: Summary
IonisationVola-
tile
ThermalSize Amount Examples
EI YesStableSmall 1-2 mg organics
CI YesStableSmall 1-2 mg organics
FAB No StableMedium0.5-1 mg
Polar/ionic organics,
organometallics, peptides,
biomolecules
FD No LabileMedium1-2 mg Non-polar organics,
organometallics
MALDI No LabileLarge 250 fmol-
500 pmol
Peptides, proteins,
polymers
ESI No labileLarge 1-300
pmol/ml
Polar/ionic organics,
peptides, proteins,
biomolecules,
organometallics, polymers
Table adapted from http://www.scs.uiuc.edu/~msweb/SLM530.pdf

22. Summary: Application ranges of various
techniques
masspec.scripps.edu/MSHistory/whatisms.php

23. Self-assessment questions
•Q1 Describe the two ion formation models in ESI
•Q2 A positive-ion ESI spectrum shows the following adjacent signals
at m/z 4348.8, 4546.5, 4762.9, 5001.0 5264.2. Calculate the
molecular mass of the molecule.
•Q3 The following m/z (979,1040,1109,1189,1280,1387,1512,1664)
were obtained by electrospray ionisation of a protein from an
aqueous solution.
–Calculate the molecular mass of the protein within 10 Da.
–Describe how the mass spectrum would have looked if the same
protein had been ionised by MALDI.
•Q4 What distance between isotopic peaks would you expect for a
+12 charge state ?
•Q5 The following peaks arose from the different isotopes
contributing to the ESI mass spectrum resulting from the protonation
of a species of relative molecular mass m to reach a charge
z.:m/z=848.40, 848.45, 848.50, 848.55, 848.60, 848.65, 848.70.
–What is the value z and what is the mass of the species giving
rise to the peak at m/z 848.55

26. Lecture 6
Tandem MS
Peptide/protein identification by MS

27. At the end of this session you should be
able to
•explain how structural information can be
obtained by Tandem MS and MALDI-TOF/PSD
•explain how mass spectrometry data can be
used to identify known and unknown
proteins

28. Tandem MS
(also termed MS
2
and MS
n
)
•Used for:
–Identify and quantify compounds in complex
mixtures
–Structure elucidation of unknown compounds
•Applied in:
–Proteomics
–Metabolomics
–Biomarker discovery
–De novo protein sequencing

29. Tandem MS
•Multistage technique:
mass selectionof precursor ion
Ion sourceMS-1
Activation and
fragmentation
MS-2
Mass analysis
of product ions
Normal
spectrum
MS/MS
spectrum

30. Tandem MS
Fragmentation techniques
•Collision-induced dissociation (CID):
–most common
–Possible with ESI coupled to triple-quad, Ion trap, FT-
ICR, and MALDI-TOF
•Electron Capture Dissociation (ECD):
–only for multiply charged biopolymers
–Primarily with FT-ICR
•Electron-Transfer Dissociation (ETD)
•Absorption of electromagnetic radiation
•Not Tandem MS, but useful fragmentation technique:
Post-source decay (PSD) combined with MALDI
reflectron TOF

31. Reflectron-TOF and Post-Source
decay for MALDI-TOF
Detector
reflectron
inducespost
sourcedecay
•(Some) parent ions fragment in field-free drift region
•Parent and product ions arrive at reflectron
simultaneously (same velocity)
•Product ions leave Reflectron earlier (smaller E
kin)
Field-free region

32. Example: MALDI-PSD TOF spectrum of a
neuropeptide
NeclaBirgül, ChristophWeise, Hans-JürgenKreienkamp and DietmarRichter, The EMBO Journal(1999) 18,5892–5900
Parent Ion

33. Simplified schematic for protein identification
from biological samples (“Proteomics”)
Cell culture
Tissue
Biofluid
Complex
protein mixture
Peptide-mass
fingerprints
Peptides
Identified proteins
Peptide
sequences
Individual or small
sets of proteins
Extraction
Separation
Cleavage
MALDI
Peptide
mass mapping
Sequencing
(LC-MS/MS)
Database
search
Database
search

34. Peptide mass mapping/
fingerprinting
•Makes use of specific cleavageagents
–Chemical cleavage: e.g. CNBr
–Digestion with endoproteases (proteolytic
enzymes): Trypsin, pepsin, chymotrypsin
etc.
–See exercises

35. Peptide mass mapping/fingerprinting
Protein
Sequence
(in database)
Theoretical
Mass Spectrum
Theoretical
Digest
QNICPRVNRIVTPCVAYGLG
RAPIAPCCRALNDLRFVNTR
NLRRAACRCLVGVVNRNPGL
RRNPRFQNIPRDCRNTFVRP
FWWRPRIQCGRIN
NTFVRPFWWRPR
IVTPCVAYGLGR
CLVGVVNR
APIAPCCR
FQNIP
...
m/z
600 900 120015001800
Abundance
Protein Peptides Mass Spectrum
m/z
600 900 120015001800
Abundance
Digest
Peptide sequences
Compare

36. De novo protein discovery
•Mass fingerprinting only practicable if
protein is already in a database
•If previously undiscovered protein: Need to
sequence
•Can be done by sequencing peptides

37. Peptide sequencing:
fragmentation rules
•Three types of bonds along backbone
•amino alkyl bond
•alkyl-carbonyl bond
•amide bond
+
NH
3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO
2
―
R
4R
1
R
2
R
3
N-terminus C-terminus

38. y
3
b
1
NH
2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO
2H
R
4R
1 R
2
R
3
z
3
c
1
x
3
a
1
Peptide sequencing:
fragmentation rules
•Each bond can be broken by fragmentation Six
possible product ions
•But: peptide bond most likely to break in low energy
CID (and MALDI/PSD):
Mostly b and y fragments

39. Peptide fragments generated by low energy
CID or PSD
N-terminus C-terminus
y
3
b
1
y
2
b
2
y
1
b
3
NH
2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO
2H
R
4R
1
R
2
R
3
m/z values of b ions: residue masses + 1 (+H
+
)
m/z values of y ions: residue masses +17 (OH
-
) + 2 (2H
+
) = 19
y
2:
+
NH
3―CH―CO―NH―CH―CO
2H
R
4R
3
b
3:NH
2―CH―CO―NH―CH―CO―NH―CH―C=O
R
1
R
2
R
3
+
For example:

40. Example: Peptide analysed by MALDI TOF
reflectron and PSD
Proposed sequence:HTH[LIN]FA[LIN]R
His
Thr
His
PheAla
Leu/
Ile/Asn
Arg
Leu/
Ile/Asn
Leu/Ile: 113.08
Asn: 114.04
y
1 y
2
y
3
y
4
y
5
y
6
y
7 y
8

41. Self-assessment questions
•Q1How are MALDI and ESI used for the
identification of proteins ?
•Q2Describe how Peptide Fingerprinting works
•Q3 A MALDI-PSD spectrum of a peptide shows
the following y-peaks:
174.8 / 288.3 / 359.0 / 506.1 / 619.6 / 756.0 / 857.6 / 995.2
Find out the masses for amino acids (e.g. at
Wikipedia). Calculate the differences between
peaks in this spectrum and suggest possible
sequences for this peptide

42. Exercises
•Exercise 1: Protein cleavage/digestion
1. Go to http://www.expasy.ch/tools/peptidecutter/
2. In the box, enter ALBU_HUMAN (this is the swissprot
name of human serum albumin) -you can also choose a
different protein if you like. Sequences and swissprot
codes can for example be found in the swissprot
database (at www.expasy.ch).
3. Scroll down, and tick the box “only the following
selection ofenzymes and chemicals”,
and then select one chemical or enzyme, which you
want to use for cleaving the albumin protein, from the list
4. Scroll back up, and click “Perform”
5. Inspect the output. How many times is albumin cleaved
by your chosen cleavage agent ? Find out what the
specificity of your cleavage agent is.

43. •Exercise 2: Calculation of molecular masses of
proteins and peptides
–1. Copy a peptide fragment from the output of
Exercise 1 (or make one up yourself), go to
http://www.expasy.ch/tools/protparam.html
–and paste your sequence into the appropriate box
(the large one).
–2. Click “Compute Parameters”.
–3. Inspect the output. What is the molecular formula of
the peptide ? How many positively and negatively
charged side-chains does your peptide have ? What
charge would it have at pH 7 ?

44. •Exercise 3. Average and monoisotopic masses
–1. Copy the molecular formula (or the one-letter code
sequence) of the peptide from exercise 2, and go to
http://education.expasy.org/student_projects/isotopident/
htdocs/
–2. Paste the formula or sequence in the appropriate box.
Make sure that you have selected the correct “Type of
composition” (e.g. “chemical formula”) in the respective
pulldown menu.
–3. Click “Submit query”.
–4. Inspect the output. How many isotopic peaks are
there? Which is the most abundant peak ? What are the
monoisotopic and the average masses ?