Download - Lecture 5
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
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
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 keV2-10 l/min
http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
Flow
Spray needle/Capillary Spray
Cone
Atmospheric pressure
Skimmers
Vacuum
To mass spectrometer
Electrospray Ionisation (ESI)
• Flowrates: 2 to 10 l/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
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
Charged amino acids
N
O
O
O-
AspartateN
OO
O-
GlutamateAcidic
3.65 4.24
N
O
N
NHHistidine
6.00
N
ON
NN
+
ArginineN
OH3N+
Lysine
Basic
10.8
12.5
Usually negatively charged at pH 7
Positively/uncharged at pH 7
Always positively charged
Charges on surface of proteins
Red: Negatively chargedBlue: Positively chargedWhite: No charge; hydrophobic
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
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 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 = 998.1 and m1 = 1060.5• n = 16, and M = 16952 Da
m/z
1n1)H](n[M
n must be an integer
nnH][M
1nn
1n
mmHm
Deconvolution of ESI mass spectra
Charge statesDeconvolutedspectrum
M = n(mn – H)
3000 30000 Mass (Da)
Quick reminder: average and monoisotopic mass
The distance between isotopic peaks reveals charge state
protein_modelingLCTmix of 6 proteins
m/z915 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/z1084 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/z500 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. KartyJonathan A. Karty
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
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+
deconvolutedraw data
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: SummaryIonisation Vola-
tileThermal Size 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 fmol-500 pmol
Peptides, proteins, polymers
ESI No labile Large 1-300 pmol/l
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
MS-2
Mass analysisof product ions
Normalspectrum
MS/MS spectrum
Tandem MSFragmentation 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
Reflectron-TOF and Post-Source decay for MALDI-TOF
D e t e c t o r
r e f l e c t r o n
i n d u c e s p o s t
s o u r c e d e c a y
• (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)
Field-free region
Example: MALDI-PSD TOF spectrum of a neuropeptide
Necla Birgül, Christoph Weise, Hans-Jürgen Kreienkamp and Dietmar Richter , The EMBO Journal (1999) 18, 5892–5900
Parent Ion
Simplified schematic for protein identification from biological samples (“Proteomics”)
Cell cultureTissueBiofluid
Complexprotein mixture
Peptide-massfingerprints
Peptides
Identified proteinsPeptide
sequences
Individual or small sets of proteins
ExtractionSeparation
Cleavage
MALDI
Peptide mass mapping
Sequencing(LC-MS/MS)
Database search
Database search
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
ProteinSequence
(in database)
TheoreticalMass Spectrum
TheoreticalDigest
QNICPRVNRIVTPCVAYGLGRAPIAPCCRALNDLRFVNTRNLRRAACRCLVGVVNRNPGLRRNPRFQNIPRDCRNTFVRPFWWRPRIQCGRIN
NTFVRPFWWRPRIVTPCVAYGLGRCLVGVVNRAPIAPCCRFQNIP...
m/z600 900 1200 1500 1800A
bundance
Protein Peptides Mass Spectrum
m/z600 900 1200 1500 1800
Abundance
Digest
Peptide sequences
Compare
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
• Three types of bonds along backbone• amino alkyl bond• alkyl-carbonyl bond• amide bond
+NH3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2―
R4R1 R2 R3
N-terminus C-terminus
y3
b1
NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H
R4R1 R2R3
z3
c1
x3
a1
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
Peptide fragments generated by low energy CID or PSD
N-terminus C-terminus
y3
b1
y2
b2
y1
b3
NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H
R4R1 R2 R3
m/z values of b ions: residue masses + 1 (+H+)m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19
y2: +NH3―CH―CO―NH―CH―CO2H
R4R3
b3: NH2―CH―CO―NH―CH―CO―NH―CH―C=O
R1 R2 R3
+
For example:
Example: Peptide analysed by MALDI TOF reflectron and PSD
Proposed sequence: HTH[LIN]FA[LIN]R
HisThrHisPheAlaLeu/
Ile/AsnArg
Leu/Ile/Asn
Leu/Ile: 113.08Asn: 114.04
y1 y2
y3
y4
y5
y6
y7 y8
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 / 995.2Find 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 ?