1.
Learning Outcomes
Students should be able to :
• Understand the overall workflow of protein identification
• Understand protein identification by Western blotting
• Understand protein identification by mass spectrometry
• Identify basic components of mass spectrometer
• Compare different ionization methods
• Compare different types of mass spectrometers

2.
• developed in Stanford in George Stark’s laboratory.
• named by W. Neal Burnette following southern blot (RNA) and
eastern blot (protein PTM) that involve protein transfer from
gel to membrane (nitrocellulose/PVDF).
• probed with Ab against protein of interest or lectins for glycans
Protein identification by
Western/Immuno Blotting
Protein transfer from gel to membrane

3.
Blocked  Wash  Primary Ab  Wash  Secondary  Wash  Develop
Skimmed PBST/TBST or lectin Ab Signal
Milk
Overview of Immuno-blotting

4.
Nitrocellulose membrane is a sticky membrane used
for immobilizing proteins in western blotting.
It can be stained with Ponceau S dye for protein
detection during western blotting.
Nitric acid is used to convert cellulose into cellulose
nitrate and water.

5.
Blocking non-specific sites
• Western blotting relies on the antibodies binding
specifically to the proteins of interest. One potential
problem is that antibodies are also proteins and would bind
to the membrane non-specifically.
• To stop this the membrane is first treated with a blocking
agent. Note that between each step in this process the
membrane is rinsed with fresh buffer to remove all
unbound traces of the previous reagent.
• One popular blocking agent is called ‘Blotto’ and contains:
5% w/v non-fat powdered milk
0.05% v/v Tween 20, in
0.05M Tris-HCl, pH 7.4, 0.2M NaCl.

6.
http://microbiollogy.blogspot.my/2014/08/diagnostic-medical-microbiology.html
Antibody Specificity in Western Blotting
Extra reading:
Choosing the right antibodies http://www.abcam.com/tag/antibody%20guide

7.
• The membrane is soaked in the specific antibody solution
and the antibodies will attach ONLY to specific site(s) on
specific protein(s). If no specific protein is present in the
sample then no antibody will attach. This provides the
specificity of the technique. This antibody is called the
primary antibody and the unattached antibody is washed
off.
• The membrane is then soaked in another antibody called
the secondary antibody. This is an antibody directed
against the first antibody. An ‘anti-antibody or antibody to
an antibody’ if you like. Again, if no primary antibody has
attached then no secondary antibody will attach. Unbound
antibody is then washed off.

8.
Types of Protein Transfer
• the aim is to completely transfer all protein bands from the
gel to the membrane
• three types of transfer systems:
a) wet system
b) semi-dry system
c) dry system
• all systems still incorporate the transfer stack, with variable
amounts of buffer/water involved
Horizontal protein
transfer
Vertical protein
transfer

9.
Wet Transfer System (Tank System)
Horizontal protein
transfer

10.
Semi-dry Transfer system- Vertical transfer
Dry Transfer
system-
Vertical transfer

11.
2D protein map Western blot
1. Visualisation based on
chemiluminescence detection
Can be followed by quantitation using densitometry

12.
2D protein map Western blot
2. Visualisation based on fluorescence
detection

13.
3. Visualisation based on chromogen/colour detection
Example of a successful
Western detection
Principle of chromogenic detection

14.
Three basic components of MS
Ion source Mass
Analyzer
Detector
Ion formation Ion separation Detection
MALDI – matrix
assisted laser
desorption
ionisation
ESI – electrospray
ionisation
Quadrupole
TOF – time of
flight
Ion Trap
Fourier
transform
Electron multiplier

15.
A series of b and y ions
are formed by
fragmentation

16.
m/z ratio (m=mass, z= charge)
Example of peptide fragment/sequence
QAEVALRCAV mass = 1059 Dalton
QAEVALRCAV-H+ mass = 1060 Da
m/z = 1060/1
= 1060
H+- QAEVALRCAV-H+ mass = 1061 Da
m/z = 1061/2
= 530.5

17.
• different proteins will give different PMF
• If the gene sequence is known, the PMF of a protein can
be derived using software

18.
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 m /z
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
a .i.
/D = /p s d _ u lf/A rg P _ C A F _ C C A _ 0 2 0 8 0 9 _ P S D _ 1 6 7 2 .8 0 _ 0 _ J 6 .F A S T /fa s t/p d a ta /1 A d m in is tra to r T u e A u g 2 7 1 3 :2 7 :4 9 2 0 0 2
A
D
S
G
E
G
D
F
I/L
A
E
G
G
G
V
R Sulf
1535.7 – 1465.3 = 70.4  Ala
1465.3 – 1350.8 = 114.5  Asp
1350.8 – 1263.5 = 87.3  Ser

19.
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 m /z
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
a .i.
/D = /p s d _ u lf/A rg P _ C A F _ C C A _ 0 2 0 8 0 9 _ P S D _ 1 6 7 2 .8 0 _ 0 _ J 6 .F A S T /fa s t/p d a ta /1 A d m in is tra to r T u e A u g 2 7 1 3 :2 7 :4 9 2 0 0 2
RVGGGEALFDGEGSDA
RVGGGEALFDGEGSD
RVGGGEALFDGEGS
RVGGGEALFDGEG
RVGGGEALFDGE
RVGGGEALFDG
RVGGGEALFD
RVGGGEALF

20.
Important parameters in MS
 Resolution – ability to distinguish between
peptides , the smallest difference in m/z that can
be detected – down to 1 Da
 Sensitivity – ability to detect even the lowest
amount of peptide, the minimum amount of
molecule require for detection – down to
femtomole level
 Accuracy – correct ID of peptides

21.
Methods of Sample Ionisation
1. MALDI
 matrix assisted laser desorption ionisation
 sample is co-crystallised with a matrix (low MM organic
matrix)
 a laser beam is used to excite the matrix (absorbs laser
wavelength (UV or IR) causing matrix-protein to expand into
gas-phase
 peptides are ionised by protonization
(addition of H+), using energy from
the laser (excited state proton transfer-
between photonized organic matrix and
a sample molecule)

22.
Good quality matrix should:
a) have the same solubility as the analyte in specific solvents
b) stable in the vacuum environment
c) prevents cluster formation
d) absorbs a desired wavelength
e) can cocrystalize the sample
f) promotes analyte ionization
Dried-droplet method: drop of aqueos matrix solution mixed
with sample,left to dry, stable for storage
Requires removal of salt and contaminants prior to sample
measurement, high content of additives such as detergents e.g
SDS can suppress ionization, resulting in no/poor spectra
acquisition

23.
2. SELDI-MS
(Surface Enhanced Laser Desorption/Ionisation MS)
Step 1 Protein (mix) spotted on a surface (chemically modified)
Step 2 Some proteins bind to the surface, others are removed by
washing
Step 3 After washing, matrix (e.g EAM) applied to the surface,
allowed to crystallize with the sample peptides
Step 4 Analyse by TOF-MS
• Binding to the SELDI surface acts as a separation step and the subset of
proteins that bind to the surface are easier to analyze
• Common surfaces include CM10 (weak-positive ion exchange), H50
(hydrophobic surface, similar to C6-C12 RP chromatography, IMAC30 (metal-
binding surface), and Q10 (strong anion exchanger)
• Surfaces can also be functionalized with antibodies, other proteins, or DNA

24.
PRINCIPLE OF SELDI-TOF CHIP ASSAYS
Source: urology.jhu.edu

25.
Advantages of SELDI:
• does not require protein electrophoresis
• only a small amount of sample (fluid, tissue) required
• robust and can be automated (HTP)
• high detection limit (femtomolar)
• allows analysis of hydrophobic proteins (membrane bound)
Example: rapid discovery of differentially expressed proteins
using femtomolar quantities of crude protein derived from
biopsy material (Lin et al. Modern Pathology, 2004)
Disadvantages:
• results are biased towards peptides and smaller proteins
(proteins <30 kDa)
• sensitivity and resolution of the TOF analyser falls off
markedly above 30kDa.

26.
3. Electrospray ionisation (ESI)
 Sample added to a solvent
 An electrical field is applied
 Produces positively charged ions in gas phase
oxidation reduction
Taylor
cone

27.
From right to left:
Delivery needle with Taylor cone  droplet formation, Coulomb
fission
(droplet explodes) with droplet evaporation  gas-phase ion
formation in transfer capillary
Taylor cone- positively charged, enriched with +ve ions due to
oxidation on the inner walls of the electroconductive
delivery needle
Nanoelectrospray
- Efficiency contributed by initial diameter of droplet formed and flow
rate
- Compared to ESI- higher sensitivity (1fmol vs 10fmol)
- smaller sprayer diameter (1-25µm vs 50-200µm)
- flow rate (1-1000nL/min vs 1-500µL/min)
- requires less voltage due to the positioning of the
delivery needle much closer to the transfer capillary

28.
 ions are accelerated in an electrical field
 flies to the detector, passing through a ‘reflectron’
 the ions are all given the same amount of energy
 thus the time-of-flight (TOF) depends on the mass
 the m/z ratio can then be calculated from the TOF
 a peptide mass fingerprint is generated
 matched against theoretically predicted tryptic peptides of all
known proteins
 excessive internal energy of ions themselves or collisions
with free gas cause dissociation immediately after the ions
generated by laser illumination exit the high speed field
region
 MALDI is often used with a TOF separator
Methods of Ion Separation (Mass Analysers)
1. TOF (Time-of-flight) Separator

29.
MALDI-TOF-TOF
Also many other hybrid machines e.g. Quadrupole-
TOF, Ion trap-TOF etc.

30.
2. Quadrupole mass analyser
 Consists of 4 circular rods
 perfectly parallel to each other
 an direct current voltage and a radio frequency voltage
are applied across the rods
 only ions of a certain m/z can travel thru the centre of the
rods at a given ratio of voltage
 other ions has unstable trajectories and will collide with
the rods
 by adjusting the voltage ratio
– an entire m/z spectrum can be scanned  PMF
– or ions with a particular m/z value can be selected

31.
3. Quadrupole ion trap mass analyzer
The same principle in both Quadrupole and Quadrupole on
trap mass analyzer. However, Quadrupole ion trap shows
higher sensitivity and ability to trap specific ions.

32.
4. Fourier-transform ion cyclotron
resonance mass spectrometry
An FTICR is essentially a cyclotron, a type of particle
accelerator in which electrons are captured in orbits by a
very strong magnetic field, while being accelerated by an
applied voltage. The cyclotron frequency is then related to
the m/z.

33.
5. Quadrupole-Time of Flight mass
analyser
Q-TOF mass spectrometer is a combination of TOF and
quadrupole instruments, a pairing that results in high mass
accuracy for precursor and product ions, strong quantitation
capability, and fragmentation experiment applicability.

34.
2D LC-MS for high throughput proteomics
• aka shotgun proteomics
• 2D gels are a bit tedious (imagine having to cut 1000
spots and do MS one by one)
• Automation  separate protein sample using
2Dimensional -HPLC
• i.e. use two types of columns to separate a complex
protein sample
• (same principle as 2D gel – separate using two
different methods e.g. charge and size)
• Each fraction is then fed sequentially into an MS
• can analyse the protein content of an entire tissue

35.
1st dimension – separate by
charge on ion exchange
column
2nd dimension – separate by
hydrophobicity on reverse
phase column

36.
Thank you