Mass spectrometry

Bionanotools and Protein Structure 2005
Mass spectrometry
of peptides and proteins
by
Henrik Karring
1

Outline
The genome and the proteome
Genomics and proteomics
An overview of the technologies of proteomics
2D gel electrophoresis
Protein visualization
Image Analysis
2

Mass spectrometry
Ionization (MALDI and Electrospray)
Mass analyzers
Detector
Protein identification by mass spectrometry
Peptide mass fingerprinting (MALDI)
Tandem mass spectrometry (MS/MS)
CAF-MS/MS
Characterization of the human cornea proteome
with reference to ocular transparency
3

What is a Genome ?
The complete DNA sequence of an organism
STATIC. The genome is the same in all cells – all the time.
4

What is a Proteome ?
A proteome is the entire protein complement
expressed by a given biological system in given
conditions.
Wilkins et al., BioTechnology 14,61-65 (1996)
DYNAMIC. The proteome is not the same in all cells and tissues.
5

What is Proteomics?
Proteomics is the systematic analysis of protein
expression patterns and functions in biological systems.
It involves identifying all proteins and determine how the
proteins interact.
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Why proteomics ?
1. Proteins are the “business end” of the genes, the final
product that carry out all the DNA instructions.
2. Proteins carry out and regulate body processes.
E.g. hormones, antibodies, enzymes, structural proteins.
3. The correlation between mRNA abundance and protein
amount is not that good.
You can have a protein in a cell/tissue without its mRNA
present.
You can have lots of mRNA without translation to protein.
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4. Alternative splicing of mRNA
5. Post-translational modifications.
Signal peptide cleavage, other proteolytic events,
phosphorylation, disulfides, cross linking etc.)
6. Protein location
(intra- or extracellular, membrane or organel associated).
7. Proteomics is used to study protein interactions
8. Most drug targets are proteins
Proteomics is the direct way to annotate the genome
8

Summary
Proteomics address fundamental problems like:
(i) does the protein exist at all ?
(ii) where is the protein ?
(what tissue, cell type, inside or outside the cell)
(iii) when is the protein present ?
(during development, inflammation, all the time….)
(iv)
Structure / Function
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2D gel electrophoresis
10

Two-dimensional gel electrophoresis
Isoelectric focusing (pI)
Molecular weight
11

Sample preparation
2D-PAGE
Cells or tissue
Extract
proteins
Good separation of proteins including
post-translational modified isoforms.
12

Step 1: the sample is applied to the immobilized pH
gradient strip over night.
13

Step 2: The isoelectric
performed.
focusing step is
Step 3: the next day the
strips are incubated in
SDS-sample buffer and
alkylated.
14

Step 4: 2-Dimensional SDS-PAGE electrophoresis
15

Protein visualization
16

Coomassie Brilliant Blue R-250 staining
MW: 826.0 g/mol
Detection limit ~ 100 ng protein
Max abs ~ 550 nm
Formula: C 45 H 44 O 7 S 2 Na

1. Binds non-specifically to protonated amino
groups by electrostatic interactions
2. Useful for densitometry
3. Simple protocol
4. Compatible with mass spectrometry

Silver staining
1. Reduction of Ag + to metallic Ag → staining
Ag + ions complexed with sulfhydryl and carboxyl
groups of proteins are selectively reduced.
1. High sensitivity
Detection limit ~ 2-5 ng protein
4. Easy to use and not expensive.
5. “Compatible with mass spectrometry”

pH 4
7
12.5%
The protein stained with
silver is not useful for
mass spectrometry
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Fluorescent dyes
1. Cydye DIGE Fluors (Cy2, Cy3, and Cy5) – cyanine
2. Cross-link to epsilon amine on lysine via amide linkage
3. Cydyes are size and charge-matched
- proteins with different labels will overlay
4. Have narrow excitation and emission bands

Internal standard (pool of 1 and 2)
Label with Cy2
Protein sample 1 (normal)
e.g. control label with Cy3
Mix labelled
samples
Image gel on Typhoon
Protein sample 2 (diseased)
e.g. treated label with Cy5
Excitation wavelength 1 Excitation wavelength 3
Excitation wavelength 2

Ettan 2D difference gel electrophoresis
(Ettan 2D-DIGE)

- no gel-to-gel variation
- high sensitivity (~ 1 ng)
- pH insensitive (no change in signal over wide pH range)
-very accurate - good statistical analysis
- can detect < 10% difference in expression
- less analysis time
- compatible with mass spectrometry

Image analysis
25

PDQuest program for 2D-PAGE
Normal Diseased Drug-treated
Compare expression profiles
Up- and down regulated
26

Functions and principles
1. Image fitting and Gaussian modeling.
2. Spot detection
3. Normalization and removing lines and speckles
4. Matching of gels
(e.g. normal, diseased, and treated tissues)
5. Analysis of data (changes in expression)
6. Annotation of spots
(linking of data to spots – intensity and MS data etc.)
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3D viewer
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Mass spectrometry
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Components of a Mass Spectrometer
High Vacuum System
Turbo pumps
Rough pumps
Inlet
Ion
source
Mass
analyzer
Detector
Recorder
HPLC
Sample plate
MALDI
Electrospray
TOF
Quadropole
Microchannel
plate
PC
Macintosh
Mass spectrometers measure the mass-to-charge (m/z)
ratios of gas phase ions
32

Ionization
M + nH + (M+nH) n+
MALDI: Matrix Assisted Laser Desorption Ionization
Sample co-crystallized with a UV-absorbing matrix
Pulsed ionization method
Produces mainly single-charged ions (+1)
ESI: ElectroSpray Ionization
Sample in solution
Continuous ionization method
Produces multiple-charged ions (+1, +2, …..)
33

What is a “matrix” for MALDI?
A couple of representative matrices that have been
demonstrated to be good "all-around" MALDI matrices.
O
O
OH
OH
HO
OH
HO
CN
2,4-dihydroxy benzoic acid
alpha-cyano-4-hydroxy
cinnamic acid
Adsorption: 337 nm
34

MALDI
Sample plate
Nitrogen UV laser (337 nm, 3 nsec pulses)
hν
1. Sample is mixed with excess
matrix and dried on a MALDI
plate.
MH +
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are ionized by
proton transfer from matrix.
+20 kV
Variable
Grid
Ground
Grid
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Target plates for MALDI
36

Electrospray Ionization
Nanoflow electrospray
Flowrate: 200-300 nL/min
37

Mass analyzers
Time-of-flight (TOF)
m/z can be calculated from the measured
time-of-flight
Quadrupole (mass filter)
ions of a certain m/z are selected
Ion trap
ion are trapped and can be further analyzed
(multiple MS/MS)

MALDI time-of-flight
Target plate (+20kV)
Reflector Detector
Reflector
Linear
Detector
Flight tube
Laser
1kV
2kV 3kV
10kV
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Reflector
A reflector is an “ion mirror” that:
1: increases the overall path
length for an ion.
2: provides higher performance
(resolution and mass accuracy).
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Quadrupole (mass filter)
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Microchannel Plate Detector (MCP)
Up close view
The channels are 12.5 microns in diameter.
(A human hair is 60 - 80 microns in diameter)
42

Signal Amplification in a
Microchannel plate
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Micromass Q-TOF Ultima Global
mass spectrometer
Peptide ions are selected in the
quadrupole
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Protein identification by
mass spectrometry
46

Peptide mass fingerprinting by
MALDI-MS
Trypsin
digestion
Abundance
p5
p3
p1
p4
p2
MALDI-MS
Peptide
extraction
and matrix
MALDI target
m/z
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Combined spectrum of sample
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Isotope cluster of peptide
Monoisotopic
Peptide
(all C 12 )
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What happens during the search?
The protein databases contain
all know protein sequences.
Search:
Which record in the
database fits the
determined masses
best ?
In-silico digestion:
The theoretical mass
of the tryptic peptides
for each protein in
the database.
Peak list
The mass of the “real” tryptic peptides
determined by the mass spectrometer
are compared to the each sequence
record in the database.
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Choose database
Choose organism
Find peak list file
Select modifications
Peptide tolerance
51

Protein score
52

Tandem mass spectrometry
Peptide fragmentation
53

Fragmentation
The most commonly observed ions are the b- and y-
ions
54

Example of MS-MS Sequencing
K EE P A N D I Q
FA
Parent ion
D
Spectrum can be complex to interpret
55

LC-MS/MS
MudPit: Multi-dimensional Protein identifiation technology
1. Digestion of complex protein sample
2. Online separation of peptides by
SCX and reverse phase
chromatography
3. MS/MS sequencing of peptides
4. Combining all MS/MS data
5. Search databases
56

Chemical-assisted fragmentation
The problems of reading sequence from MS/MS spectra can be
encountered by the use of CAF
Lysine modification
Prevents the reaction of
lysine residues in
second step
57

Sulfonation
Sulfonation of N-terminus.
Introduces a negative charge
58

Charges on sulfonated ion
Tryptic peptide:
Sulfonated peptide:
-
+
Lys/Arg
+
Lys/Arg
Peptide ion in positive mode:
-
+
+
Lys/Arg
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After quadrupole selection
Parent ion
60

After applying collision energy
R
R
R
R
R
Parent ion
-
CAF-ELINSWVESQTNGIIR + S
N
I
L
E
CAF
(R)
I/L
113
I/L
113
G
57
N
114
E
T Q S 129
101 127 87
V W S
99 186 86
N
I/L
114 113
I/L
E
113 129
134
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Something about sensitivity
(a 50 kDa protein is used in the examples)
500 pico mol -1 nano mol (25-50 micro g)
SDS-PAGE (Coomassie): 20 pico mol (1 micro g)
SDS-PAGE (Silver): 0.2 - 2 pico mol (10 - 100 nano g)
Silver is 10 -100 x times more sensitive than Coomassie
Edman degradation: 10-20 pico mol (0.5 - 1 micro g)
Amino acid analysis: 10-20 pico mol (0.5 - 1 micro g)
Sensitivity of MALDI or Electrospray is in
the femto mol range or into even atto
mole amount. 10 -6 micro
10 -9 nano
10 -12 pico
10 -15 fento
10 -18 atto
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The End
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