The document discusses proteomics, which is the study of the entire complement of proteins in a cell or organism. It defines key proteomics terms like proteome and describes techniques used in proteomics like protein separation, 2D gel electrophoresis, mass spectrometry, and protein digestion. The goals of proteomics include detecting and comparing protein expression profiles to understand biological processes and discover drug targets. Proteomics provides important insights not available through genomics alone.

1. BY MUHAMMED RASHID AK
M.pharm pharmaceutics

2. What is a Proteome?
• The terms proteomics and proteome
were coined by Wilkins et al. in 1994
to describe the entire collection of
proteins encoded by genomes in the
human organism.

3. • Proteomics differs from protein chemistry at
this point since it focuses on multiprotein
systems rather than individual proteins and
uses partial sequence analysis with the aid
of databases.

4. Why is the Proteome Important?
• It is the proteins within the cell that:
– Provide structure
– Produce energy
– Allow communication
– Allow movement
– Allow reproduction
• Proteins provide the structural and
functional framework of cellular life

5. What is Proteomics?
• Proteomics refers to the systematic
analysis of protein profiles of entire
cells, tissues, organisms, or species.
• It represents the protein counterpart to
the analysis of gene function.

6. • Proteomics is an attempt to describe or
explain biological state and qualitative
and quantitative changes of protein
content of cells and extracellular biological
materials under different conditions to
further understand biological processes.

7. Aims of Proteomics
• Detect the different proteins expressed by
tissue, cell culture, or organism using
various techniques.
• Store those information in a database
• Compare expression profiles between a
healthy cell vs. a diseased cell
• The data comparison can then be used for
testing and rational drug design.

8. Proteomics vs Genomics
• DNA sequence does not predict if the protein is in an
active form
• RNA quantitation does not always reflect corresponding
protein levels
• Multiple proteins can be obtained from each gene
(alternative splicing)
• Genomics cannot predict post-translational modifications
and the effects thereof
• DNA/RNA analysis cannot predict the amount of a gene
product made (if and when)

9. Proteomics and genomics are inter-dependent
Genome Sequence
Proteomics
Genomics
mRNA Protein Fractionation
Primary Protein products
2-D Electrophoresis
Functional protein products
Proteomics
Protein Post-Translational
Identification Modification
Determination of gene

10. Why is Proteomics Important?
• Identification of proteins in normal and disease
conditions
– Investigating epidemiology and taxonomy of pathogens
– Analysis of drug resistance
• Identification of pathogenic mechanisms
– Reveals gene regulation events involved in disease
progression
• Promise in novel drug discovery via analysis of
clinically relevant molecular events
• Contributes to understanding of gene function

11. Proteomic Methodologies
• Analysis of protein expression patterns
• Analysis of protein Sequence
Information
• Analysis of protein structure/function
relationships

12. Proteomic Methodologies
• Complex protein mixtures are separated by
2-D gel electrophoresis
• Then individual proteins are isolated from
spots and are identified by using mass
spectrometry
• Individual proteins are sequenced, followed
by database searches
• Bioinformatics

13. Schematic representation of basic
proteomics analysis
Protein mixture
separation
proteins
digestion
peptides
MS analysis

14. MS data
Software assisted database search
peptide sequence identification

15. Protein separation
• First step in proteomic research
• The biological sample for analysis is first
pulverized; homogenized, sonicated, or
disrupted to form a mixture containing cells
and sub cellular components in a buffer
system

16. Proteins are extracted from this mixture
using
• Detergents: SDS, CHAPS
help membrane proteins to dissolve and
separate from lipids.
• Reductants: DTT, thiourea
reduce disulfide bonds or prevent oxidation
• Denaturing agents: urea, acids
alter the ionic strength, pH of the solution
and destroy protein–protein interactions,
disrupting secondary and tertiary structures

17. • Enzymes: DNAse, RNAse , Digestion is
achieved by enzymes. Protease inhibitors
are often used to prevent proteolytic
degradation.

18. Extracted proteins are separated by
following techniques
• 1D-SDS-PAGE (1-dimensional sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis)
• 2D-SDS-PAGE (2-dimensional sodium
dodecyl sulfate-polyacrylamide gel
• electrophoresis)
• IEF (isoelectric focusing)

19. • HPLC (high performance liquid
chromatography)
• Size exclusion chromatography
• Ion exchange chromatography
• Affinity chromatography

20. Gel Electrophoresis
• Motion of charged molecules in an electric field.
• Polyacrylamide gel provides a porous matrix
– (PAGE – Polyacrylamide Gel Electrophoresis)
• Sample is stained with comassie blue to make it
visible in the gel.
• Sample placed in wells on the gel

21. 1-D Gel Electrophoresis
• Separation in only 1 dimension: size.
• Smaller molecules travel further through the
gel then large molecules, thus separation.

22. Steps
• 1. Preparation of a loading buffer
containing a thiol reductant (e.g., DTT)d
SDS
• 2. Dissolving protein in the loading buffer
• 3. Binding of SDS to protein to form a
protein-SDS complex
• 4. Applying to the gel
• 5. Applying high electric voltage to the
ends of the gel
• 6. Migration of the protein-SDS complex
• 7. Formation of bands on the gel in order of

23. 2DE
• most effective way of separating proteins
• Help to identify diseases-specific proteins,
drug targets, indicators of drug efficacy and
toxicity.
• separation of post-translationally modified
protein from the parent one is usually
achieved by 2DE.
• Several thousands of different proteins can
be separated from each other in one gel.

24. • 2-DE separation is conducted based on
the electrical charge and molecular weight
(size) of the proteins
• First step is to separate based on charge or
isoelectric point, called isoelectric focusing.
• Then separate based on size (SDS-PAGE).

26. steps 2-DE are
1. Preparation of the sample
2. Solubilization
3. Reduction
4. IPG-IEF
5. Equilibration
6. SDS-PAGE

27. Preparation of the Samples for 2-DE
• The method with minimum modification
should be chosen, otherwise artifactual
spots may form on the gel and mislead the
operator
• Serum, plasma, urine, cerebrospinal fluid
(CSF), and aqueous extracts of cells and
tissues are often require no pretreatment.
• They can be directly analyzed by 2-DE
following a solubilization step with a
suitable buffer (e.g., mostly phosphate
buffered saline, or PBS).

28. • Liquid samples with low protein
concentrations or large amounts of salt
should be desalted and concentrated prior to
2-DE.
• Desalination can be achieved by dialysis or
liquid chromatography

29. Solubilization
• In order to avoid misleading spots on the 2-
DE profile and to remove salts,
lipids,polysaccharides, or nucleic acids
interfering with separation,
• solubilization procedure involves disruption
of all noncovalently bond protein
complexes into a solution of polypeptides

30. Reduction of Proteins
• involves reduction of disulfide bonds in the
protein samples.
• DTT or β-mercaptoethanol are the most
widely used reducing agents.
• noncharged reducing agents (e.g., tributyl
phosphine: TBP*) have been preferred
recently.

31. ISOELECTRIC FOCUSING (IEF)
• The isoelectric point is the pH at which the
net charge of the protein molecule is neutral
• Proteins in mixture are separated based on
their isoelectric points (pI) following the
voltage application.
• With the commercially available IEF
apparatus, proteins can be separated into
12–20 fractions.

32. • Equilibration step:
• . gels are often equilibrated prior to
second dimension analysis in order to allow
separated proteins to interact with SDS.
• This interaction will provide migration
during SDS-PAGE analysis.
• Equilibration can be achieved by
incubating the strips for 15 minutes in 50
mM Tris buffer of pH 8.8 in the presence of
SDS, DTT,urea, and glycerol

33. SDS-PAGE
• Second Dimension.
• Separation by size.
• Run perpendicular to Isoelectric focusing.
• The only unresolved proteins after the first
and second dimensions are those proteins
with the same size and same charge – rare!

35. • 2D-PAGE Analysis
• Gel matching, or “registration”, is the
process of aligning two images to
compensate for warp.

36. List of 2-D GEL DATABASES
• One can find an extensive list of such
databases by following these links.
• We would discuss a few “Interesting ones”.
• World 2-D PAGE
• NCIFCRF
• DEAMBULUM-Protein Databases
• Ludwig Institute of Cancer Research
• Phoretix

37. • LINKS
• Z3 system (Compugen) -
http://www.2dgels.com/
• Melanie3 (SIB) -
http://us.expasy.org/melanie/
• ProteomWeaver (Definiens) -
http://www.proteomweaver.com/
• PDQuest (Bio-Rad) -
http://www.biorad.com/
• Delta2d (Decodon) -
http://www.decodon.com/

38. PROTEIN DIGESTION
reasons behind this approach
• MS instruments used for the analysis of
separated proteins run for peptides with
fewer errors.
• Because the greater the mass of the protein,
the greater the possibility of obtaining
inaccurate results.
• difficult to perform MS on very large and
hydrophobic proteins.
• Sensitivity of mass measurements of
peptides is superior to that of proteins.

39. • enzymes used for digestion.
• Proteases are the most widely used
enzymes.
Trypsin is the most frequently used serine
protease
• Glu-C, so-called V8-proteases, is an endoprotease
digesting proteins at carboxyl side of
glutamate residues in the buffer solutions
• Nonspecific proteases such as subtilysin,
pepsin, proteinase K, or pronase are also
used in proteomics

40. • Cyanogenbromide (CNBr) is the most
widely used chemical digestion
agent. It cleaves proteins at methionine
residues

41. Identification of separated protein
• Second step in proteomic study.
• The basic identification process is analysis
of the sequence or mass of six amino acids
unique in the proteome of an organism, then
to match it in a database.

42. MASS SPECTROMETRY (MS) FOR
PROTEOMICS
• The ion producing source
• A mass analyzer: converts components of a
mixture into ions based on their
mass/charge ratio (m/z ratio)
• A detector to detect the resolved ions.
• most frequently used instruments of MS-
based proteome analysis are

43.  MALDI-TOFF
matrix-assisted laser desorption ionization
time of flight.
• MALDI refers to the source of ionization
whereas TOF indicates type of the mass
analyzer.
 ESI Tandem Ms
• electrospray ionization mass spectrometry
performed in multistage
• based on the production of multiply charged

44.  Matrix-assisted laser desorption/ionization
(MALDI)
 Electrospray ionization (ESI)
• The resultant ion is propelled into a mass
analyzer by charge repulsion in an electric
field.
• Ions are then resolved according to their
m/z ratio.
• Information is collected by a detector and
transferred to a computer for analysis

45. TECHNIQUES USED FOR
STRUCTURAL PROTEOMICS
• aims the determination of three-dimensional
protein structures in order to better
understand the relationship between protein
sequence, structure,and function
• NMR and x-ray crystallography are used t
determine the structure of macromolecule
• To obtain optimal results, protein should
possess minimum 95% purity.
• .

46. • the molecule under investigation should be
purified by gel or column separation,
dialysis, differential centrifugation, salting
out, or HPLC prior to structural analysis.

47. X-ray crystallography
• X-ray crystallography is used to determine
the tertiary structure of a protein
• Much information about flexibility of
protein structure has also come from x-ray
• crystallography data. the production of
crystals for x-ray studies can sometimes
cause structural anomalies. They might
mask native architectural features.
• membrane proteins are not readily
amenable to existing crystallization methods

48. NMR
• NMR measures proteins in their native state
• Precise crystallization, which is often
difficult, is not necessary for conducting
structural analysis by NMR
• NMR is increasingly being recognized as a
valuable tool, not only in three-dimensional
structure determination, but also for the
screening process
• Proteins with large molecular weight (up to
30 kDa) can be analyzed

49. The most significant advantages of NMR
spectroscopy are
• it reveals details about specific sites of
protein molecules without a need to solve
the whole structure.
• It is sensitive to motions of most chemical
events which in turn provides direct and
indirect examination of motions within
micro-time scale (milliseconds to
nanoseconds, respectively).

50. Differential Protieomics