Proteomics

recent advances

1. PROTEOMICS
Sqn Ldr Raka Maitra
Guide: Lt Col Harsh Kumar

2. Introduction
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3. Nomenclatures
Genome – sum total of all the genes in an
organism.
Genomics – study of sequence of all genes in
an organism.
Transcriptome – sum total of all the RNA
transcripts an organism can make in its lifetime.
Transcriptomics – study of levels of RNA
produced from many genes in a cell at a given
time.

4. Proteomics - Definitions
Proteome – properties and activities of all the
proteins that an organism makes in its lifetime.
PROTEin complement to a genOME .
Proteomics - the qualitative and quantitative
comparison of proteomes under different
conditions to unravel biological processes.

5. Proteomics - Definition
Proteomics is a scientific discipline concerned
with systematic analysis of proteins present in
cells at a given time under given conditions.
Proteomics includes the identification,
characterization and quantitation of the entire
complement of proteins in cells, tissues or
whole organisms with a view to understanding
their function in relation to the life of the cell.

6. Protein Synthesis

7. Why do we need Proteomics?
Level of transcription of a gene ¹ Level of
expression of the gene
mRNA - degraded rapidly
- translated inefficiently
Post-translational Modifications /
Translocations
One gene / transcript > many proteins
One protein > many processes
One function > many proteins

8. Genome Vs Proteome
Information stored in the genome is used
differently in different cells
Multigenic diseases
Incorrect modification of a normal protein
Diagnosis of disease
Targets for drugs

9. Protein Structure
Primary structure

10. Protein Structure
Secondary structure

11. Protein Structure
Tertiary structure

12. Protein Structure
Quaternary structure

13. Technologies in Proteomics
Protein separation
Protein detection
Protein analysis
Identification
Measurement of activity

14. Protein Separation
2-D polyacrylamide gel electrophoresis (2-D
PAGE) :
separation by charge (isoelectric point)
separation by mass
HPLC
Dialysis
Differential Centrifugation
Salting Out

15. 2-D PAGE

16. Protein Detection
Staining – non-quantitative
Coomassie blue
Silver
SYPRO Ruby Red
Fluorescence - quantitative
Autoradiography – more sensitive quantitation

17. Comparison
Of
Different
Protein
Detection
Methods

18. Mass Spectrometry
Separates proteins according to their mass-to-
charge (m/z) ratio
Ionization of proteins – ions propelled towards the
analyzer by electric field - resolves each ion according
to its m/z ratio ® detector ® computer for analysis
Ionization methods :
Matrix-assisted laser desorption/ ionization (MALDI)
Electrospray ionization (ESI)

19. MALDI-TOF
Ionization by MALDI –
protein suspended in a crystalline matrix
laser energy causes rapid excitation of matrix
passage of matrix and analyte ions into gas phase
ionized protein accelerated by electrostatic field and
expelled into a flight tube
Time-of-flight analyzer (TOF) –
when accelerated by application of a constant
voltage, the velocity with which an ion reaches the
detector is determined by its mass

20. Electrospray Ionization (ESI)
Production of gaseous ions by application of a
potential to a flowing liquid resulting in the formation of
a spray of small droplets with solvent-containing
analyte
Solvent is removed from the droplet by heat or
collision with a gas
Droplet size further decreases to become unstable
and explode into even finer droplets
Electrostatic repulsion is sufficiently high to cause
desorption of the analyte ions
Passed to the mass spectrometer

21. Mass
Spectrometric
Separation
Of
Different
Proteins

22. Protein Analysis
Methods:
Structural Analysis
X-ray Crystallography
Nuclear Magnetic Resonance
Post-translational Analysis – activity based
analysis
Newer techniques – analysis of proteins in complex
mixtures without separation

23. X-ray Crystallography
Crystallization:
Supersaturation
Snap freezing
Factors affecting:
pH
Temperature
Precipitant used – salts / polyethylene glycol
Protein concentration

24. X-ray Crystallography
Process:
X-rays directed at crystal of protein / derivative of the
protein containing a heavy metal atom
Rays scattered in pattern dependent on electron
densities in different portions of the protein
Images translated into electron density maps
Superimposed on one another manually or by
specialized computer programs
Construction of a model of the protein

25. X-ray Crystallography
Disadvantages:
Time consuming
Expensive
Requires very specialized training and equipment
Advantages:
Reveals very precise and critical structural data
about amino acid orientation
Used to understand protein interactions

26. NMR Spectroscopy
Nuclear dipoles in the sample align in a magnetic field
Transmitter pulses radio waves to the sample
Hydrogen nuclei absorb energy and ‘flip’ from one
orientation to another
Later flip back and readmit that energy as radio
signals
Nuclei in different chemical environments on
molecules radiate different energies
Amplified by a receiver and stored on a computer
Software routines interpret the chemical environments

27. NMR Spectroscopy
Crystallization is not necessary
Facility to reveal details about specific sites of
molecules without having to solve their entire structure
Sensitivity to motions on time scale of most chemical
events
Adept at revealing how active sites of enzymes work
Transfer Nuclear Overhauser Spectroscopy
(TrNOESY) facilitates shape determination of small
molecules bound to very large ones, and helps define
the binding pocket of the macromolecule.

28. Post-translational Analysis
Phosphoproteins
Sample digested by proteolytic enzyme alone vs
proteolytic enzyme + phosphatase
Phosphoantibodies to precipitate phosphorylated
proteins before mass spectrometry
Stains to detect phosphoproteins in polyacrylamide
gels
n-linked sugars
Use of glycosylases

29. Protein Microarrays
Used for:
protein purification
expression profiling
protein interaction profiling
Steps:
Capture
Washing
Uncoupling
Analysis

30. Newer Techniques
Phage Display:
Creation of peptide or protein libraries on viral
surfaces
Peptides or proteins remain associated with their
corresponding genes
Cloning of Ligand Targets (COLT)
Alternative to phage display
small peptide sequences bind to larger domain units
within proteins
Used to discover new domains and new proteins

31. Bioinformatics
Building and manipulation of biological
databases.
Integration of mathematical, statistical and computer
methods to analyze biological, biochemical and
biophysical data.
Databases of DNA sequences of genomes. Eg.
Genbank, EMBL
Collections of proteomics databases for organisms.
Eg. Swissprot, Flybase
Database of computationally derived protein
structures.

32. Proteomics in Life Sciences
Proteomics has many diverse practical
applications in the fields of:
Medicine
Biotechnology
Food sciences
Agriculture
Animal genetics and horticulture
Environmental surveillance

Pollution

33. Applications in Medicine
•Protein changes during normal processes like
differentiation, development and ageing
•Abnormal protein expression in disease development
(especially suited for studies of diseases of multigenic
origin)
•Diagnosis
•Prognosis

34. Applications-contd.
•Identification of novel drug targets
•Selection of candidate drugs
•Surrogate markers
•Targets for gene therapy
•Toxicology
•Mechanism of drug action

35. Applications
Understanding gene function
Understanding the molecular regulation of the cell
Identification of multiprotein complexes
Studying cellular dynamics and organization
Studying macromolecular interactions

36. Type II Diabetes at the
Molecular Level
Aim: Human skeletal muscle is being analysed to find
proteins whose expression correlates with the
development of T2D.
Project design: Comparison of healthy and diabetic
persons of normal and obese build.
Sample treatment: Punch biopsies are collected
and snap frozen or rapidly transferred to CPA for
labelling with [
35
S]-methionine.

37. T2D
Results so far: Several markers for T2D
development have been identified and patented. Post
translational Modifications play a decisive role in the
development of the disease.
Significance: Modulation of the expression of these
proteins, this might offer a new treatment for diabetes.

38. Mechanism Behind
Rheumatoid Arthritis
Aim: Human synovial fluid (including the cells
therein), the surrounding tissues and serum are being
analysed with the aim to identify pathophysiological
changes that can be used diagnostically or
therapeutically.
Project design: Comparison of synovial fluids,
biopsies and sera from persons at different stages
during the development of arthritis.
Sample treatment: Biopsies and cells are collected
from synovial fluid, labelled with [
35
S]-methionine and
tested with sera from arthritic patients.

39. RA
Results so far: This approach has allowed us to
identify early antigens and antibodies in the synovial
fluids.
Significance: Early results suggest that this may
allow us to determine the effectiveness of treatment.

40. Changes That Occur During
Ageing
Aim: Human skin biopsies are being studied to reveal
changes in the physical structure of skin in order to
chart the changes that occur with age so we will be
able to develop treatments which will retard the
process or protect it from environmental stress.
Project design: Comparison of protein expression
patterns in human skin biopsies from persons at
different ages, different sites on the body and of
different gender.
Sample treatment: Skin biopsies are collected,
labelled with [
35
S]-methionine.

41. Ageing
Results so far: An extensive database is being built
up and some markers have already been identified.
Significance: Ageing is something that no one can
avoid. Therefore, these results have applications not
only in the cosmetic industry, but also in many other
fields, because our skin is very active. Eg. in the
excretion of waste products; the regulation of
temperature; the protection from harmful radiation;
and the uptake of certain types of medication.

42. Colon cancer
Aim: Proteomics is also being used here to carry out
a search for molecular markers, which could predict
prognosis from pre-malignant to malignant disease
and predict efficacy of cytotoxic therapy in a reliable
way.
Project design: Human biopsies of colorectal tissue
are collected at different stages of cancer
development and compared to identify progression
markers.
Sample treatment: Colorectal tissue biopsies are
labelled with [
35
S]-methionine.

43. Colon cancer
Results so far: We have developed procedures by
which bacterial infections can be avoided so that this
does not influence the analysis of the polyps. A
number of surprisingly large changes have been
selected and the proteins identified.
Significance: There are no reliable methods that can
be used in predicting the response of patients to
radiation or chemotherapy. Considering the increasing
number of persons affected and the treatment options
available, a convenient non-invasive diagnostic kit
would be of great value.

44. Pathogenesis of
Cholesteatoma
Aim: Testing the two theories: whether the
hyperkeratinization of this destructive middle-ear
disease is due to changes in the lipid metabolism or
whether it is due to bacterial infection or both. If it is
the former, the goal is to identify which metabolic
pathways have failed, and if the latter, the goal is to
determine which microorganisms are present, and
whether they are the direct or indirect causative
agents or only opportunistic infections.
Project design: Biopsies are collected and divided into
the pathologically distinct parts of the epithelium and
compared against normal skin from the ear canal.

45. Cholesteatoma
Sample treatment: The various parts are labelled
with [
35
S]-methionine.
Results so far: A number of striking differences has
been identified which suggest that bacteria are not
directly involved in the aetiology of the disease.
Significance: Direct treatment would spare the
patients for surgical intervention.

46. Free Radicals in Ischaemia
and Thrombosis
Aim: Characterization of the damage caused by free
radicals in human blood following for example acute
disorders like ischaemia, or chronic disorders like
vasoconstriction, and their role in the development of
thrombi. The ultimate goal is to find points at which the
process could be regulated or the detrimental effects
alleviated.
Project design: The effect of anoxia and reperfusion
will be investigated on isolated blood vessels and cells
to follow the development of oxidative damage. This
project will then compare the effects seen in man with
those seen in an animal model.

47. Free Radicals
Sample treatment: A combination of fluorescent
labelling and [
35
S]-methionine labelling will be used
depending upon the type of sample. All samples will
be analysed by 2DGE.
Results so far: Reaction pattern of the granulocyte
has been intensively studied and several reaction
pathways have been characterised. Resistance
arteries have been studied, and proteins whose
expression correlates to hypertension have been
identified.
Significance: Cardiovascular diseases are one of the
major reasons for mortality in the developed World.

48. Drug Proteomics
Unrecognized connections between proteins and
protein complexes, drugs, and biological processes
are identified with proprietary proteomics technologies.
Ability to select druggable targets, choose lead
molecules with key features, and reject targets with
safety concerns.
Rational framework to elucidate mechanism of action
for bioactive molecules.
Bioinformatics - translating experimental data into
mechanistic models of cellular function and
dysfunction and ways to interfere using compounds
and drugs.

49. Environmental Proteomics
Studies of the health effects of environmental agents.
Many environmental chemicals interact directly with
cellular protein to modify their functions and
interactions.
Environmental agents also may affect gene
expression and the levels of protein products of those
genes.
Proteomics technologies used to investigate the
interplay of environmental agents and the proteome.

50. Microbial Proteomics
Bacterial genomes encode all possible virulence
determinants, vaccine candidates, and potential drug
targets.
A completed genomic sequence allows high
throughput analysis of the proteome.
Mycoplasma pneumoniae - second smallest genome
of any self- replicating life form and encodes 679
putative proteins.
Genome- predicted proteins correlated with those
actually present, detecting any biological event that
generates a protein of different molecular composition
than that predicted.

51. Recent Advances
Reverse Proteomics
Starting point is the DNA sequence of the genome
Transcriptome and proteome are predicted in silico
This information is used to generate reagents for
their analysis.
Shotgun Proteomics
Complete bypassing of  2D-gel electrophoresis
Enabled by Multidimensional Protein Identification
Technology (MudPIT).

52. Conclusion