1. Condensed phase ionisation techniques: spray methods
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
Spray needle/
Capillary
Ions
Spray
To mass spectrometer
Flow
Cone
Nebuliser gas
Skimmers
Corona discharge
Vacuum
Atmospheric pressure

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
Spray needle/Capillary
Spray
To mass spectrometer
Flow
3-4 keV
2-10 ml/min
Cone
Skimmers
Atmospheric pressure
Vacuum

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. Ionisation models Ion evaporation model
Spray needle tip
Multiply charged droplet
Ion evaporation model
Spray generates multiply charged droplets
Solvent evaporation leads to increasing charge density
When charge density too high: Coulombic explosion
Rayleigh limit is reached
Analyte molecule
Multiply charged droplet
Charged residue model

11. Charged amino acids Usually negatively charged at pH 7
Aspartate
Glutamate
Acidic
3.65
4.24
Usually negatively charged at pH 7
6.00 O
Positively/uncharged at pH 7 N N NH
Histidine N O +
Arginine
H3N
Lysine
Basic
10.8
12.5
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+
13+
12+
11+
10+ 9+ 8+ 7+ 6+
Charge state series
750
1000
1250
1500
1750
2000
2250
2500
2750
m/z

14. How to determine the molecular mass of a protein from
an ESI-MS spectrum
Observed ions have composition [M+nH]n+
Let m1,m2,…, mn : m/z values of the different peaks
mn =
Its neighbouring peak to the left:
mn+1 =
Solving both equations for n and M:
n =
M = n(mn – H)
e.g. m2 = and m1 =
n = 16, and M = Da
848.7
808.3
771.6
893.3
738.1
942.8
707.4 1 n
1)H] (n [M +
998.1
1060.5
m/z
n must be an integer

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

16. Quick reminder: average and monoisotopic mass

17. The distance between isotopic peaks reveals charge state +1
1.00 +4
0.25
0.51 +2
Jonathan A. Karty

18. Charge States and distance between isotopic peaks
1695.7
1696.2
1696.7
m/z
0.0
0.5
1.0
1.5
a.i.
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 Ca2+
excess Ca2+
raw data
deconvoluted

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
Ionisation
Vola-tile
Thermal
Size
Amount
Examples EI
Yes
Stable
Small
1-2 mg
organics CI
FAB No
Medium
0.5-1 mg
Polar/ionic organics, organometallics, peptides, biomolecules FD
Labile
Non-polar organics, organometallics
MALDI
Large
250 fmol-500 pmol
Peptides, proteins, polymers
ESI
labile
1-300 pmol/ml
Polar/ionic organics, peptides, proteins, biomolecules, organometallics, polymers
Table adapted from

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 , , , 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, , , , , ,
What is the value z and what is the mass of the species giving rise to the peak at m/z

26. Tandem MS Peptide/protein identification by MS
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 MS2 and MSn)
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: Ion source MS-1
Activation and fragmentation
MS-2
Mass analysis
of product ions
mass selection of precursor ion
MS/MS spectrum
Normal
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
Field-free region
(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 Ekin)

32. Example: MALDI-PSD TOF spectrum of a neuropeptide
Parent Ion
Necla Birgül, Christoph Weise, Hans-Jürgen Kreienkamp and Dietmar Richter , The EMBO Journal (1999) 18, 5892–5900

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

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

35. Peptide mass mapping/fingerprinting
Protein
 Peptides
Mass Spectrum
Digest
Abundance
600
900
1200
1500
1800
m/z
Compare
Protein
Sequence
(in database)
Theoretical
Mass Spectrum
Peptide sequences
Theoretical
Digest
QNICPRVNRIVTPCVAYGLGRAPIAPCCRALNDLRFVNTRNLRRAACRCLVGVVNRNPGLRRNPRFQNIPRDCRNTFVRPFWWRPRIQCGRIN
NTFVRPFWWRPR
IVTPCVAYGLGR
CLVGVVNR
APIAPCCR
FQNIP
...
Abundance
600
900
1200
1500
1800
m/z

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
+NH3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2―
N-terminus
C-terminus
Three types of bonds along backbone
amino alkyl bond
alkyl-carbonyl bond
amide bond

38. Peptide sequencing: fragmentation rulesb1 c1 R1 R3 R2 R4
NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H x3 y3 z3
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
NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H
N-terminus C-terminus y3 y2 y1
For example: R1 R2 R3
b3: NH2―CH―CO―NH―CH―CO―NH―CH―C=O + R3 R4
y2: +NH3―CH―CO―NH―CH―CO2H
m/z values of b ions: residue masses + 1 (+H+)
m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19

40. Example: Peptide analysed by MALDI TOF reflectron and PSD
His
Leu/
Ile/Asn
His
Thr
Leu/
Ile/Asn
Ala
Phe
Arg
Leu/Ile:
Asn:
 Proposed sequence: HTH[LIN]FA[LIN]R

41. Self-assessment questions
Q1 How are MALDI and ESI used for the identification of proteins ?
Q2 Describe how Peptide Fingerprinting works
Q3 A MALDI-PSD spectrum of a peptide shows the following y-peaks: / / / / / / / 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
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
3. Scroll down, and tick the box “only the following selection of enzymes 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
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
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 ?