6. Our Latest Proteomics Research
For full publication list visit my Research gate account
https://www.researchgate.net/profile/Khalid_Hakeem
OR
Personal website
www.Khalidhakeem.weebly.com
Omics (IF=2.77) ABAB (IF=1.89) Mol. Breeding
(IF=3.25)
ABAB (IF=1.89) IJMR (IF=2.47)
7. Contents
•Introduction
•Techniques involved
•Protein isolation
•Sample preparation
•First dimension: Isoelectric focusing
•Second dimension: SDS-PAGE
•Detection of protein spots: staining
•Imaging analysis & 2D Gel databases
•Spot handling: excision, in gel digestion
•Mass spectrometry
•Software analysis
•Applications
•Challenges
8. Science 291 (2001) 1221.
WHAT IS PROTEOMICS?
The analysis of complete
complements of proteins:
identification and quantification;
modifications; interactions; and
activities. FUNCTION.
AND HOW DO THESE CHANGE
DURING A BIOLOGICAL
RESPONSE?
Peck SC (2005) Update on Proteomics in Arabidopsis. Where do we go from
here? Plant Physiol 138: 591-599
9. Major Proteomics Directions
Adapted from Human Proteome Organization (www.HUPO.org)
PTMs
Rose et al.(2004) Plant J 39: 715-733.
“Proteomics is an
increasingly
ambiguous term
being applied to
almost any aspect of
protein expression,
structure or function.”
10. Tools of Functional Genomics
Colebatch et al (2002) Functional Genomics: tools of the trade. New Phytol 153: 27-36.
11. •Whole Genome Sequence –complete, but does not
show how proteins function or biological processes
occur
•Post-translational modification –proteins sometimes
chemically modified or regulated after synthesis
•Proteins fold into specific 3-D structures which
determine function
•Gain insight into alternative splicing
•Aids in genome annotation
Why Proteomics ?
12. Solving the Puzzle of Protein Function
Proteomics is a
multipotent tool central
to research efforts in
many fields and
disciplines. Maximum
functional utility will
come from joint efforts.
13. A Single Gene Can Produce Many Proteins
Peck (2005) Plant Physiol 138: 591
Targeting
sequence
Principle:
One gene ≠ one transcript ≠
one protein
ONE Genome but MANY
Proteomes!
14. Proteomics and genomics are inter-dependent
Genome Sequence
mRNA
Primary Protein products
Functional protein products
Determination of gene
Genomics
Proteomics
Proteomics
Protein Fractionation
2-D Electrophoresis
Protein
Identification
Post-Translational
Modification
15. MALDI-TOF @ Indian Institute of Integrative Medicine ,Jammu.
( Applied Biosystems ,Model 4800 )
Mascot search engine
(http://www.matrixscience.com)
Basic Proteomic Analysis Scheme
Protein
Mixture
Individual
Proteins
Blast &
Mascot analysis
Protein
identification
16. Protein extraction and sample preparation
Normal Stressed
Reduction and alkylation
SDS-PAGE (2nd
Dimension), Visualization (Coomassie BB/Silver staining)
HPLC/Mass spectrometry and protein identification
Isoelectric focusing (1st
Dimension)3…pH…10+ - + -3…pH…10
3…pH…10(kDa) M
100
30
10
3…pH…10(kDa) M
100
30
10
LS-MS peptide-mass fingerprint spectrum of the tryptic digest of differential protein and its
idenification on MASCOT
absent
Over-expressed
induced
under-expressed
Spot numbering (1, 2, 3….x), excision and digestion
Plant Material
Proteinextraction,resolutionanddigestionProteinidentification
17. The goal of two-dimensional electrophoresis is to separate and
display all gene products present.
* It is the only method currently available which is capable of
simultaneously separating thousands of proteins.
* The first dimension of 2-DE
- isoelectric focusing (IEF).
- pH gradient support
-pI
* TheSecond dimension of 2-DE
- SDS as surfactant.
- Molecular mass.
•High resolution from independent protein parameters.
*In the early 1970s, first use of 2-DE to separate serum proteins.
The second dimension of 2-DE - sodium dodecyl
sulfate PAGE (SDS-PAGE).
18.
19. •A critical step in 2-DE.
Solubilization, denaturation, reduction & removal of non-protein sample
components.
Optional modifications for more proteins displayed, shorter focusing
time, and more sharply focused spots:
- Chaotropes: 8M Urea
2M Thiourea & 7M Urea
- Surfactants: 4% CHAPS
2% CHAPS & 2% Sulfobetaine 3-10
- Reducing agents: 100mM DTT
2mM TBP (Tributyl phosphine) &/or 65mM DTT
Sample preparation
20. Separating the Proteome
• The protein genome is separated by several different
methods.
• Many researchers are first separating portions of the
genome, such as isolating organelles, and then
analyzing that portion.
• This is because often proteins of interest, regulatory
proteins are in low abundance.
• The most commonly used method is 2-dimensional
gel electrophoresis.
– Consists of using isoelectric focusing with SDS
polyacrylamide gel electrophoresis
21. Isoelectric focusing
• This separates proteins based on isoelectric point
• The isoelectric point is the pHat which the protein has no net charge.
• pHgradients may be large 2-10 orsmall 6-7 (Optimum=4-7)
• Typically this is done with an immobilized pHgradient gel strip orwith a
tube gel containing a low concentration of polyacrylamide.
• Ampholytes are added to create a pHgradient in an electric field and the
proteins are loaded.
• The IEF gel is placed in an electrophoresis systemforup to 24 hours
and the proteins formtight bands at theirisoelectric point.
• The IEF gels are now ready forthe second method.
22. Figure is from “Principles of Biochemistry” Lehninger, Fourth Edition
23. SDS Polyacrylamide Gel Electrophoresis
• The second dimension separates the proteins based on size.
• There are two parts, the stacking gel which concentrates the
sample and the running gel that is used to separate the
proteins.
• The IEF gel is soaked in a solution containing chemical to
denature the proteins including sodium dodecyl sulfate (a
detergent which gives the proteins a net negative charge).
This means that all proteins will move in one direction.
• The IEF gel is then put in the one long well in the stacking
gel, sealed in place with agarose, and the proteins subjected
to an electric field to separate.
• The larger proteins are found at the top and the smaller
ones are found at the bottom of the gel.
24. 2-Dimensional Gel Electrophoresis
• In a 2D gel the proteins appear as spots on the gel
rather than bands. These spots can then be further
processed or used for mass spectrometry directly.
• Further processing usually includes spot excision,
trypsin digestion, and mass spectromety
• Analysis may also include differential 2D gel
electrophoresis
– In this case a control and sample are separately
labeled with a fluorescent molecule.
– The samples are mixed and electrophoresed in the
same gels.
– A laser scanner is used to identify each spot and a
program puts the two images together.
26. Visualization of proteins
Coomassie blue staining
Detect 36-47ng
Silver staining
Detect 0.5-1.2ng
Fluorescent staining
Detect 1-2 ng
From Jefferies, et al.,
http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201
Images from
http://www.kendricklabs.com/2d+CoomassieBlue.htm
http://www.unil.ch/dbcm/page48211_fr.html
27.
28. Alternate Separation Methods
• The first dimension is run in larger
agarose tube gels with ampholytes.
– This has less resolution than
polyacrylamide gels. The tubes are
sliced and the proteins are allowed
to diffuse out.
• Gel regions are cut, proteins eluted
and the proteins are then separated
by capillary electrophoresis.
• Capillary electrophoresis has a much
greater resolution for the proteins
mass.
29. Alternative Separation Methods
• Whole proteome is analyzed at once.
• Proteome is digested with protease (trypsin)
• Digested proteome is injected to HPLC with
2 columns in series (mixed bed ion exchange
and reverse phase)
• Peptides are eluted from ion exchange onto
reverse phase and then separated on
reverse phase column.
• Peptides then enter ESI-MS-MS
36. Mass Spectrometery
• Separates ions based on mass to charge ratio.
– Charges are placed on the protein or the peptide by
ionization.
• Two most common types of ionization are:
• Matrix-Assisted Laser Desorption Ionization.
– MALDI causes fragmentation of the protein during
ionization. Can be used to get more information
about the fragments. Easier to do than ESI.
• Electrospray ionization (ESI)
– ESI can give whole protein masses as well as complex
masses. If the proteins is first separated by reverse
phase HPLC before injection only the subunits
masses will be known.
37. Matrix-Assisted Laser Desorption Ionization
(MALDI)
• MALDI causes fragmentation of the protein
during ionization.
• It Can be used to get more information
about the fragments. Easier to do than ESI.
• Requires sample to be placed in matrix that
absorbs appropriate wavelength light.
• Matrix generates heat and forms ions of
matrix and what is around it.
38.
39. “MALDI-TOF” mass spec
• Matrix-Assisted:
Your favorite
molecule is co-
crystallized (dried)
with a light-
adsorbing
compound
(“matrix”).
40. “MALDI-TOF” mass spec
• Laser
Desorption/Ionizatio
n: A pulse of laser
light is used to force
molecules into gas
phase and ionize
them.
+
+ +
+ ++ +
42. MALDI-TOF mass
spectrometry
…and allowed to drift
down a long high-
vacuum flight tube.
• At the end of the
tube, ions strike a
detector.
• Drift time (time of
flight) is measured
by sophisticated
electronics.
+
+ + ++
+ +
(-)
47. Mass Analyzers
• Important parameters
– Sensitivity
• How few ions can be detected.
– Resolution
• How well different masses can be determined.
– mass accuracy
• How reproducible and correct are the masses.
48.
49.
50.
51. Interpreting a mass spectrum
• x-axis is…
–tof
–m / z
–“mass” of ion
• y-axis is…
–“hits” on the
detector
–number of ions
detected 365.0 760.6 1156.2 1551.8 1947.4 2343.0
Mass(m/z)
0
4.5E+4
0
10
20
30
40
50
60
70
80
90
100
%Intensity
Spec#1MC[BP=1053.6,44590]
1053.6285
656.1051
869.4992
1117.6282
1550.87361072.6033 1355.7562549.3439 822.4886
1426.8657 1690.9995
617.4088
1566.8917
916.5234 1036.5975
1890.0891
591.3327 741.4249 1569.9111
893.4636585.3485 1075.6256
2008.27261443.80271278.6373679.4092537.3699 1088.6108919.5099 1674.6634800.4719 1436.7851 1893.11101252.6669 2033.17691003.8684 1625.0525 2185.28701432.1174 1856.6127527.2609
53. 2D-PAGE Analysis Software
• 2D-PAGE technology has been in use for over 20 years,
and potentially provides a vast amount of information
about a protein sample.
• However, due to difficulties with data analysis, it remains
only partially exploited.
54. Analysis problems
• It can be very difficult to compare the results of two
experiments to yield a differential expression profile:
• Can be severe warping of gel due to
– uneven coolant flow
– voltage leaks
– tears in gel
• Can be problems with normalisation of
– background
– spot intensity
• Can be differences in sample preparations.
55.
56. Current state of software
• Correct identification and alignment of spots from the two
gels has generally been a process with a lot of manual
intervention - hence very slow.
• The processing power available with today’s PCs means
that automated analysis is starting to become possible.
• One vendor claims a throughput of 4 gel pairs per hour
can be compared and annotated by an experienced user
of their package.
57. Automated gel matching
• Gel matching, or “registration”, is the process of
aligning two images to compensate for warp.
• Some packages still require the user to identify
corresponding spots to help with gel matching.
• The Z3 program from Compugen has a fully-
automated gel matching algorithm:
– define set of small, unique rectangles.
– compute optimal local transformations for rectangles.
– Interpolate to make smooth global transformation.
• Note that this makes use of spot shape, streaks,
smears and background structure, which other
programs discard.
58.
59.
60.
61. Spot detection
• Once the gel images have
been matched, the program
automatically detects spots.
Algorithms are generally
based on Gaussian
statistics.
62. Spot Quantitation
• The positions of detected spots are calibrated to give
a pI / mW pair for each protein.
• A value for the expression level of the protein can be
calculated from the overall spot intensity.
• Some programs do not quantitate each gel separately,
but calculate relative intensity pixel by pixel. This may be
a more accurate approach.
63. Differential Expression
• The user can set threshold
values for the detection of
differential expression. This
helps reduce the amount of
information displayed at
once.
• In this example, a protein
expressed only in the
second sample is circled in
red. The yellow circles show
proteins which are
differentially expressed.
64. Annotation
• Some systems allow semi-automatic annotation of spots,
based on a database of proteins listing their pI / mW
values.
• Proteins of interest can also be excised from the gel and
sent on to mass spectrometry for definitive identification.
The ProteomeWorks system from Bio-rad offers such an
integrated solution for 2D-PAGE and MALDI.
65. Multi-experiment Analysis
• One useful feature of modern programs is the ability to
collate data from many runs of the same experiment.
• Spots which only appear in one gel are likely to be
artifacts, and are removed from the analysis.
• This is an excellent way to reduce noise and enhance
weak signals.
67. Applications of Proteomics
• Protein Profile- A Global view of proteins of Interest
• Differential display- Biomarker discovery
• Protein post translational modification- Protein Function
68.
69. Biological Applications
• Proteome maps
– starting point for major study in genomics
– questions of interest:
– How much of the genome is transcribed and translated in the living
organism?
– What effect different growth conditions have on the proteome?
• studies are going on in
– Eukaryotes like Humans
• extensively modify their proteins by N- or C-terminal cleavages
• decorate them with sugars and/or phosphates, sulfates... PTMs
• Yeast Saccharomyces cerevisiae, ....
• Fruit fly Drosophila melanogaster, ....
• Plant Arabidopsis thaliana, ....
70. Biological Applications, cont.
• Tracking complexity
– host-pathogen or host-parasite interactions
• nitrogen fixation in legumes by association with bacteria
(Rhizobium) to form nodules
• infection of flax by flax rust
• Immunogetic proteins
– identifying proteins from infectious disease agents
recognised by the immune system
– vaccine candidate for microbial pathogens (e.g. Chlamydia
trachomatis infection)
– allergy research: which grass pollens are most
immunogenic?
– identification of allergens (proteins) in Latex (gloves) by a 2D
PAGE run using Latex as a sample!
71. • Improved agricultural products
– engineering resistance to pathogens/parasites into various plants
– most of these resistance mechanisms involve expression of toxic or
protective proteins
– discovery of new toxic or protective proteins
– wool proteome project to investigate economically important characters
like colour and fibre strength
• Value added agricultural products
– remanufacture low value products
• proteinaceous whey as a by-product of cheese manufacture
• investigation whether this whey can be used to grow recombinant
bacteria for biotechnological production
Biological Applications, cont.
72. • Quality control
– Is the hamburger mince sold as beef really beef or a mixture
of beef and kangaroo or even buffalo?
– proteome technology brings precision and definition to a new
level in protein-based products
• For further applications please refer to the ProXPRESS PIP
on the intranet
– forensic sciences
– microbiology
– epidemiology
– taxonomy
Biological Applications, cont.
73. Proteomics contribute to Target Identification and
Validation
HUMAN
GENOME
DISEASE
ASSOCIATIONS
PUTATIVE
TARGETS
TARGET
FUNCTION
IN CELL
BIOLOGICALLY
VALIDATED
TARGETS
HIT
IDENTIFICATION
HITS TO
LEADS
LEAD
OPTIMISATION
TOXICOLOGY
EFFICACY/ P.O.C. DEVELOPMENT
Knowledge of direct interactions
partners, location, expression &
modifications helps describe a
protein’s function within the cell
Network of interactions and
pathway expression predicts
toxicology, required drug
characteristics and side effects.
74. Toxicology and Pharmaceuticals
• Multiple overlapping pathways are influenced by toxins or drug
treatment
– simultaneous identification, characterization and
quantification of numerous of gene products and their PTMs
– massively parallel approach offered by Proteomics
• Retinoic acid
– used in dermatology and onco-haematology
– retinoic acid acylation of proteins (PTM)
– detection of this protein retinoylation with proteomics
• Phosphorylation
– “on” or “off” signals of biochemical pathways by
kinases and phosphatases, complex networks
• etc....
75. Cancer
• Carcinogenic products act similarly to
pharmaceutical agents, affecting the PTMs and
the level of expression of numerous proteins
– oncogene product alterations & cell cycle specific
protein modifications play important role in
tumorgenesis and cancer progression
• studies are going on in
– brain, thyroid, breast, lung, colon, kidney, bladder,
ovary, bone marrow
76. Rice genotypes (G) 1 2 3 4 5 6 7 8 9 10 ----------------------------------20
Assays for N-use efficiency
G6
cv. Rai Sudha
High NUE
G8
cv. Munga Phool
Low NUE
Proteomics analysis for differential NUE
N N N N
N-use efficient proteins
qRT- PCR conformation
Working out the Nitrogen Efficient mechanism in rice and wheat
genotypes using proteomics
77. Cadmium toxicity induced alterations in the root proteome of green gramin
contrasting response towards iron supplement
Results suggest that green gram plants exposed to cadmium stress are able to
change the nutrient metabolic balance in roots, but in the mean time regulates
cadmium toxicity by iron supplements.
4 pI 7
14.4
21.5
31.0
45.0
66.2
97.4
MK
+Fe/-Cd
1
23
4
5
67
8
9
10
1112
13
14
15
16
17
18
19
20
21
22
2
3
A
+Fe/+Cd
D
-Fe/-Cd
C
14.4
21.5
31.0
45.0
66.2
97.4
MK
4 pI 7
-Fe/+Cd
B
8
15
14
19
12
17
9
18
10
11
202
3
221 2
3 13
1
2
3
7
8
9
10
1112
13
14
15
18
19
20
21
22
2
3
1
2
3
67
8
9
10
1112
13
14
15
16
17
18
19
20
21
22
2
3
4
5
Sowbiya Muneer†
*, Khalid Rehman Hakeem†
*, Rozi Mohamed and Jeong Hyun Lee.Int. J. Mol.
Sci.2014,15, 1-x manuscripts doi:10.3390/ijms150x000x
78. On going Proteomics projects @ UPM
•Mapping the proteome of thick-walled and rapidly
growing bamboo for the development of thick walled
bamboo plantlets. 2014-15 (118,000 RM)…..Continue
•Proteomic identification of gaharu systnesis enzymes
in pathogen-induced Aquilaria for the production of
high-impact compounds. (340,000 RM)…….Submitted
•Plant diversity, Ecosystem Dynamics and Proteomics
of Matang Mangrove Forest, Taiping, Perak. (300,000
RM)…….Submitted
79. Challenges for 2D Technology
• Whole cell extracts are too complex.
How can complexity be reduced?
• Abundant & soluble proteins are easily
characterized.How can rare and
membrane proteins be found?
• Large format gels are difficult to handle
and slow. Is there an alternative?
80. Challenges for 2D Technology
• Abundant & soluble proteins are easily
characterized.How can rare and
membrane proteins be found?
•
• Increase sensitivity by fluorescent dye
labelling! Use ProXPRESS for gel
imaging!
81. Challenges remain with Gels
• Gels & staining limit S/N, dynamic range
– today’s standard staining methods
• silver staining: 10 to 70 -200 ng/spot (enough for MS)
• Coomassie Blue: less sensitive
• fluorescent dyes expectations: 0.1 ng to 10 mg/spot (necessary
for faint protein amounts)
• Gels can be selective
• Membrane proteins need special conditions
• Low copy number proteins are missed
• fluorescent dyes expectations: 0.1 ng to 10 mg/spot (necessary
for faint protein amounts)
