Proteomics

1. PROTEOMICS
BY
HANIF ALFAVIAN

2. DEFINITION
• Study set of proteins that particular time, under particular circumstances, in
biological place such as, cells , tissues or an organism is called proteome.
• The word proteome is a blend of protein and genome, and was coined by Marc
Wilkins in 1994 while working on the concept as a PhD student.
• The proteome is the entire set of proteins , produced or modified by an organism or
system.

3. THE CHALLENGES OF PROTEOMICS
• Splice variants create an enormous diversity of proteins
• ~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins
• Splice variants may have very diverse functions
• Proteins expressed in an organism will vary according to age, health, tissue, and
environmental condtion
• Proteomics requires a broader range of technologies than genomics

4. REGULATION OF THE HUMAN
GENOME
Alternative splicing
Allows for multiple proteins from one gene..
Also uses different splice sites within introns
GTACCGATTGTAGG….AGGGGCTAG
Exon 1 Exon 2 Exon 3 Exon 4
Isoform 1
Isoform 2
Isoform 3

5. DIVERSITY OF FUNCTION IN SPLICE
VARIANTS
• Example: the calcitonin gene
• Gene variant #1
• Protein: calcitonin
• Function: increases calcium uptake in bones
• Gene variant #2
• Protein: calcitonin gene-related polypeptide
• Function: causes blood vessels to dilate

6. POST-TRANSLATIONAL
MODIFICATIONS
• Post-translational modifications are defined as any changes to the covalent bonds
of a protein after it has been fully translated.
• Addition of chemical groups to one or more amino acids on the protein

7. CHEMICAL MODIFICATIONS
• Phosphorylation: activation and inactivation of enzymes
• Acetylation: protein stability, used in histones
• Methylation: regulation of gene expression
• Acylation: membrane tethering, targeting
• Glycosylation: cell–cell recognition, signaling
• GPI anchor: membrane tethering
• Hydroxyproline: protein stability, ligand interactions
• Sulfation: protein–protein and ligand interactions
• Disulfide-bond formation: protein stability
• Deamidation: protein–protein and ligand interactions
• Pyroglutamic acid: protein stability
• Ubiquitination: destruction signal
• Nitration of tyrosine: inflammation

8. PRACTICAL APPLICATIONS
• Comparison of protein expression in diseased and normal tissues
• Likely to reveal new drug targets
• Today ~500 drug targets
• Estimates of possible drug targets: 10,000–20,000
• Protein expression signatures associated with drug toxicity
• To make clinical trials more efficient
• To make drug treatments more effective

9. TECHNOLOGIES FOR PROTEOMICS
• 2-D gel electrophoresis (2-dimensional)
• Separates proteins in a mixture on the basis of their molecular weight and
charge
• Mass spectrometry
• Reveals identity of proteins based on computer software that can uniquely
identify individual proteins
• Protein chips
• A wide variety of identification methods
• structure, biochemical activity, and interactions with other proteins
• Yeast two-hybrid method
• Determines how proteins interact with each other

11. SEPARATION AND ISOLATION OF
PROTEINS
• One dimensional gel electrophoresis
• Isoelectric Focusing= IEF
• Two dimensional gel electrophoresis
• High Performance-Pressure Liquid
• Affinity Chromatography
• Size Exclusion Chromatography
• Ion Exchange Chromatography

12. ONE DIMENSIONAL GEL
ELECTROPHORESIS
• The work:
1 - Prepare Loading buffer containing sodium dodecyl sulfate and a thiol reducing
agent.
2 - Solving the Protein Loading Buffer
3 - binding proteins and sodium dodecyl sulfate complex formation
4 - complexes incorporating sodium dodecyl sulfate - protein into the gel
5 - establish an electric potential between the Jelly Oatmeal
6 - Migration complex
7 - bonds (bond) based on molecular weight proteins

13. ISOELECTRIC FOCUSING

15. 2-D GEL ELECTROPHORESIS
• Polyacrylamide gel
• Voltage across both axes
• pH gradient along first axis
neutralizes charged proteins at
different places
• pH constant on a second axis
where proteins are separated by
weight
• x–y position of proteins on
stained gel uniquely identifies the
proteins

16. CAVEATS ASSOCIATED WITH 2-D
GELS
• Poor performance of 2-D gels for the following:
• Very large proteins
• Very small proteins
• Membrane-bound proteins

17. STAINING
One of the oldest methods of stained proteins is Coomassie Blue
Disadvantage: Extract large amounts protein.
Stained with silver nitrate and a more general approach is better because it is more
sensitive.
Disadvantage: In some cases, this color interact with characteristics of proteins
Labeled Before performing the first dimension electrophoresis with radioactive
proteins in vitro.
Disadvantage: In some cases the staining severely answers

18. LIMITATIONS OF TWO-
DIMENSIONAL ELECTROPHORESIS
• It is time-consuming and laborious
• The method like PCR for proteins is not accessible and therefore not able to detect
proteins with low copy numbers (low expression)

19. IDENTIFICATION OF PROTEINS
• In many cases, knowing the isoelectric point and molecular weight proteins are not
adequate for identification, because in some cases, two or more different proteins
may have a similar molecular weight and isoelectric.
• Edman
• Mass Spectrometer

21. EDMAN
• Amine end the desired protein to be sequenced
• Alternatively the desired protein from the gel isolated and using enzymatic
digestion, peptide fragments were sequenced by Admen.
• Disadvantage : large Protein is required and In the end, more than 50% protein,
amino end blocked and can not be sequencing.

22. MASS SPECTROMETER
• Proteins Partial sequencing and protein identification
• MS technique enables us to get information construct the proteins or peptides, such
as MW and amino acid sequences.
• This information can be used to search nucleotide databases to identify the protein
can be used.

23. MASS SPECTROMETRY
• Measures mass-to-charge ratios
of ions
• Components of mass
spectrometer
• Ion source
• Mass analyzer
• Ion detector
• Data acquisition unit

25. ION SOURCES USED FOR
PROTEOMICS• Proteomics requires
specialized ion sources
• Electrospray Ionization
(ESI)
• With capillary
electrophoresis and
liquid
chromatography
• Matrix-assisted laser
desorption/ionization
(MALDI)
• Extracts ions from
sample surface
ESI
MALDI

26. MASS ANALYZERS USED FOR
PROTEOMICS
• Ion trap
• Captures ions on the
basis of mass-to-
charge ratio
• Often used with ESI
• Time of flight (TOF)
• Time for accelerated
ion to reach detector
indicates mass-to-
charge ratio
• Frequently used with
MALDI
Ion Trap
Time of Flight
Detector

27. A MASS SPECTRUM

28. IDENTIFYING PROTEINS WITH MASS
SPECTROMETRY
• Preparation of protein sample
• Extraction from a 2-D gel
• Digestion by proteases — e.g., trypsin
• Mass spectrometer measures mass-charge ratio of peptide fragments
• Identified peptides are compared with database
• Software used to generate theoretical peptide mass fingerprint (PMF) for
all proteins in database
• Match of experimental readout to database PMF allows researchers to
identify the protein

29. LIMITATIONS OF MASS
SPECTROMETRY
• Not very good at identifying minute quantities of protein
• Trouble dealing with phosphorylated proteins
• Doesn’t provide concentrations of proteins
• Are only able to identify hundreds of proteins in a single day

30. PROTEIN CHIPS
• Thousands of proteins
analyzed
simultaneously
• Wide variety of assays
• Antibody–antigen
• Enzyme–substrate
• Protein–small
molecule
• Protein–nucleic acid
• Protein–protein
• Protein–lipid
Yeast proteins detected
using antibodies

31. FABRICATING PROTEIN CHIPS: PHYSICAL ARRAY THAT CAN
HOLD PROTEINS, ISOLATE THEM FROM EACH OTHER, AND
PREVENT THEM FROM BECOMING DENATURED
• Protein substrates:
minipads
• Polyacrylamide or
agarose gels
• Glass
• Nanowells
• Proteins deposited on
chip surface by robots
Polydimethylsiloxane

32. READING OUT RESULTS
• Fluorescence
• Most common method
• Fluorescent probe or tag
• Can be read out using standard nucleic acid
microarray technology
• Surface-enhanced laser desorption/ionization
(SELDI)
• Laser ionizes proteins captured by chip
• Mass spectrometer analyzes peptide fragments

33. DIFFICULTIES IN DESIGNING PROTEIN
CHIPS
• Unique process is necessary for constructing each
probe element
• Challenging to produce and purify each protein
on chip
• Proteins can be hydrophobic or hydrophilic
• Difficult to design a chip that can detect both
• Protein’s function may be dependent on
posttranslational modification or an interaction
with another biological molecule

34. REGULATION OF TRANSCRIPTION
TATA boxUE
Gene expression requires the
following:
A DNA-binding domain
An activation domain
A basic transcription apparatus

35. YEAST TWO-HYBRID METHOD
• Goal: Determine how proteins interact with each
other
• Method
• Use yeast transcription factors
• Gene expression requires the following:
• A DNA-binding domain
• An activation domain
• A basic transcription apparatus
• Attach protein1 to DNA-binding domain (bait)
• Attach protein2 to activation domain (prey)
• Reporter gene expressed only if protein1 and
protein2 interact with each other

36. A SCHEMATIC OF THE YEAST TWO-HYBRID
METHOD

37. HUMAN INTERACTOME (NATURE
2005)

38. DATABASE BANK
•
1. Profound(http://prowl.rockefeller.edu)
•
2. MASCOT(www.matrixscience.com)
•
3.pubmed
•
4. Expasy (www.expasy.ch)
•
5.Swiss-Prot (www.ebi.ac.uk)
•
6.IPI (International Protein Index)
•
7.SWISS-2DPAGE database

39. EXPASY SITE

40. MEDICAL APPLICATIONS OF
PROTEOMICS
• 1 - Cancer Research
• 2 - Infectious Diseases
• 3 - Heart disease

41. BIOMARKER AND CLINICAL
APPLICATIONS OF PROTEOMICS
• Molecules that show Physiological changes in the cell or tissue or organism and
compared to the control sample the simplest definition
The biomarker.
• Biomarker is biochemical molecules that are able to show a patient and able to
measured.
• Biomarker can identify disease progression or treatment effect

42. BIOMARKER FEATURES
• Show main feature of the disease, especially in the early stages of the disease.
• In comparison with Abnormalities related to the disease are sufficient specificity and
sensitivity.
• At Laboratory tests can able for identify and trust.
• it is noninvasive and relatively easy and inexpensive it is to show.

43. APPLICATION
• Disease Cardiac – Vascular and MI
• Schizophrenia
• Alzheimer's
• Progression of neurodegenerative diseases such as PD and AD
• autoimmune Patients
• Sickle cell disease(pr in membrane of RBC)
• MS disease

44. FUTURE PROSPECTS
• The next decade may see the complete deciphering of the
proteome of yeast (done already)
• More initiatives, like the Human Liver Proteome Project, are
underway
• Better understanding of disease: prognosis and diagnosis