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Lecture 5
Condensed phase ionisation
techniques: spray methods
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
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)
Condensed phase ionisation
techniques (2): spray methods
Overview
• Thermospray
• APCI: Atmospheric pressure chemical
ionisation
• APPI: Atmospheric pressure
photoionisation
• Electrospray ionisation
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
Atmospheric pressure
Vacuum
www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
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
2-10 ml/min
3-4 keV
Cone
Skimmers
Atmospheric pressure
Vacuum
http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
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….
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
What it looks like
Ionisation models
Ion evaporation model
1. Spray generates multiply
charged droplets
2. Solvent evaporation leads to
increasing charge density
3. When charge density too
high: Coulombic explosion
Spray
needle tip
Multiply charged
droplet
Analyte
molecule
Rayleigh limit
is reached
Multiply charged
droplet
www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
Charged residue model
Charged amino acids
Usually negatively charged at pH 7
O-
4.24 O
O
3.65
O
N
Acidic
N
Aspartate
Glutamate
O
6.00
N
Positively/uncharged at pH 7
O
O-
N
NH
Histidine
Basic
Always positively charged
12.5
+N
+
O
H3N
10.8
Lysine
N
O
N
N
Arginine
N
Charges on surface
of proteins
Red: Negatively charged
Blue: Positively charged
White: No charge;
hydrophobic
Examples
• ESI of large molecules usually produces multiply
charged ions
• e.g. proteins:
12+
11+
10+
9+
13+
8+
Charge state series
7+
6+
750
1000
1250
1500
1750
2000
2250
2500
2750
m/z
• Each peak corresponds to the same protein, but
with different number of protons attached:
Observed ions are [M+nH]n+
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
848.7
808.3
771.6
893.3
• mn =
738.1
942.8
[M+ nH]
n
• Its neighbouring peak to the left:
707.4
998.1
1060.5
• mn+1 =
[M+ (n+1)H]
n+1
• Solving both equations for n and M:
m/z
n must be an integer
mn+1  H
• n=
mn  mn+1
• M = n(mn – H)
• e.g. m2 = 998.1 and m1 = 1060.5
• n = 16, and M = 16952 Da
Deconvolution of ESI mass spectra
Deconvoluted
spectrum
Charge states
M = n(mn – H)
3000
30000
Mass (Da)
Quick reminder: average and
monoisotopic mass
The distance between isotopic
peaks reveals charge state
mix of 6 proteins
LCT
prot_mix_0724a 651 (10.856) Sm (SG, 2x6.00); Cm (648:651)
protein_modeling
505.3506
TOF MS ES+
783
505.3506
100
+1
%
1.00
506.3584
mix of 6 proteins
LCT
915.4818
prot_mix_0724a 350 (5.837) Sm (SG, 2x6.00); Cm (343:374)
100
506.3584
protein_modeling
915.7363
TOF MS ES+
1.86e3
915.7363
507.3566
915.4818
507.3566
915.9765
+4
%
915.9765
915.2247
0
500
m/z
501
502
503
504
505
506
507
508
509
510
511
512
916.2311
915.2274
916.2311
916.4857
0.25
916.4857
916.7402
0
915
916
917
m/z
mix of 6 proteins
918
prot_mix_0724a 655 (10.923) Sm (SG, 2x6.00); Cm (645:675)
LCT
protein_modeling
1086.5515
TOF MS ES+
454
1086.5515
100
1086.0433
1086.0433
0.51 1086.0444
+2
%
1087.0444
1087.5529
1087.5529
1088.0460
Jonathan A. Karty
1088.0460
0
m/z
1084
1085
1086
1087
1088
1089
1090
Charge States and distance between
isotopic peaks
a.i.
1.5
0.1
+10
1.0
0.5
0.0
1695.7
1696.2
1696.7
m/z
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
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 !
Ionsiation techniques: Summary
Ionisation
Vola- Thermal Size
tile
Amount
Examples
EI
Yes
Stable
Small
1-2 mg
organics
CI
Yes
Stable
Small
1-2 mg
organics
FAB
No
Stable
Medium
0.5-1 mg
Polar/ionic organics,
organometallics, peptides,
biomolecules
FD
No
Labile
Medium
1-2 mg
Non-polar organics,
organometallics
MALDI
No
Labile
Large
250 fmol500 pmol
Peptides, proteins,
polymers
ESI
No
labile
Large
1-300
pmol/ml
Polar/ionic organics,
peptides, proteins,
biomolecules,
organometallics, polymers
Table adapted from http://www.scs.uiuc.edu/~msweb/SLM530.pdf
Summary: Application ranges of various
techniques
masspec.scripps.edu/MSHistory/whatisms.php
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
Lecture 6
Tandem MS
Peptide/protein identification by MS
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
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
Tandem MS
• Multistage technique:
mass selection of precursor ion
Ion source
MS-1
Activation and
fragmentation
Mass analysis
of product ions
MS-2
Normal
spectrum
MS/MS
spectrum
Tandem MS
Fragmentation techniques
• Collision-induced dissociation (CID):
– most common
– Possible with ESI coupled to triple-quad, Ion trap, FTICR, 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
Reflectron-TOF and Post-Source
decay for MALDI-TOF
in d u c e s
p o s t
Field-free region
s o u rc e d e c a y
D e te c to r
r e fle c tr o n
• (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)
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
Simplified schematic for protein identification
from biological samples (“Proteomics”)
Cell culture
Tissue
Biofluid
Peptide-mass
fingerprints
Extraction
Complex
protein mixture
Separation
Peptide
mass mapping
Cleavage
Peptides
MALDI
Database
search
Identified proteins
Individual or small
sets of proteins
Sequencing
(LC-MS/MS)
Database
search
Peptide
sequences
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
Peptide mass mapping/fingerprinting
 Peptides
Mass Spectrum
Abundance
Protein
Digest
600
900
1200
1500
1800
m/z
Compare
QNICPRVNRIVTPCVAYGLG
RAPIAPCCRALNDLRFVNTR
NLRRAACRCLVGVVNRNPGL
RRNPRFQNIPRDCRNTFVRP
FWWRPRIQCGRIN
Peptide sequences
Theoretical
Digest
NTFVRPFWWRPR
IVTPCVAYGLGR
CLVGVVNR
APIAPCCR
FQNIP
...
Theoretical
Mass Spectrum
Abundance
Protein
Sequence
(in database)
600
900
1200
m/z
1500
1800
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
Peptide sequencing:
fragmentation rules
R1
R2
R3
R4
+NH ―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO ―
3
2
N-terminus
•
•
•
•
Three types of bonds along backbone
amino alkyl bond
alkyl-carbonyl bond
amide bond
C-terminus
Peptide sequencing:
fragmentation rules
a1
b1
c1
R1
R2
R3
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
Peptide fragments generated by low energy
CID or PSD
b1
b2
b3
R3
R2
R1
R4
NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H
N-terminus
C-terminus
y3
y2
y1
For example:
R1
R3
R2
+
b3: NH2―CH―CO―NH―CH―CO―NH―CH―C=O
R3
y2:
R4
+NH ―CH―CO―NH―CH―CO H
3
2
m/z values of b ions: residue masses + 1 (+H+)
m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19
Example: Peptide analysed by MALDI TOF
reflectron and PSD
y7
y8
y6
y5
y4
y1
Arg
y2
Leu/
Ile/Asn
Leu/Ile: 113.08
Asn: 114.04
Ala
y3
Phe
Leu/
Ile/Asn
His
Thr
His
 Proposed sequence: HTH[LIN]FA[LIN]R
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:
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
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 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.
• 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 ?
• 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 ?