4. Proteome is a defines the complete set of
proteins expressed during a cell’s entire
lifetime.
Proteomics is the study of the proteome; it
uses technologies ranging from genetic
analysis to mass spectrometry.
Proteomics assesses activities, modifications,
localization, and interactions of proteins in
complexes.
6. Proteome indicates the total proteins expressed by a
genome in a cell or tissue.
Biomarkers detection might allow identification of
patients who would benefit from further evaluation.
With the development of proteomic techniques,
proteome analysis provides a fast, non-invasive
diagnostic tool for patients with various diseases.
The advent of highly sensitive proteomic technologies
can identify proteins associated with development of
diseases well before any clinically identifiable alteration.
MS has a high resolving power and identifies proteins
with more accuracy.
7.
8. 1 the behavior of gene products is difficult or
impossible to predict from gene sequence.
2 Even if gene is transcribed, its expression may be
regulated at the level of translation.
9. 1. Structural Proteomics:- The ultimate aim of this
proteomics is to build a body of structural information
that will help predict the probable structure and potential
function for almost any protein from knowledge of its
coding sequence.
2. Functional proteomics:- It refers to the use of
proteomics techniques to analyze the characteristics of
molecular protein-networks involved in a living cell.
3. Expression proteomics:- It refers to the quantitative
study of protein expression between sample differing by
some variable.
10. Protein/peptide separation.
Identification and characterization of resolved proteins
by MS.
Data analysis and applications.
11. Primary structure- is sequence of specific amino acid
in polypeptide chain.
Secondary structure- the primary polypeptide chain
gets properly folded in the form of alpha- helix, beta
pleated sheet, random coils and turns.
12. Tertiary structure- secondary structure interact with
each other chemically to form the 3 dimensional
shape of the proteins.
Quaternary structure- interaction between different
polypeptide unit.
13.
14. Separation of proteins
One dimentional electrophoressis
2 D electrophossis(modern)
Multi-dimensional HPLC (modern)
Analysis of proteins
Mass Spectrometry (modern)
Database utilization
15. 1. Sample collection, handling and storage.
2. Separation of individual proteins by 2-D
electrophoresis.
3. Protein characterization.
4. Identification by mass spectrometry or other
methods.
5. Storage, manipulation, and comparison of the
data using bioinformatics.
16. Gel based
– SDS-PAGE
– 2-DE
Off gel base
– LC (SCX, RP-LC, Immuno affinity)
Quantitative proteomics
– iTRAQ, ICAT, SILAC
MS
– MALDI, LC-MS, CE-MS
17.
18. The large-scale analysis of protein
Protein structural comparisons can help to
identify the function of newly discovered
genes
X-ray crystallography, NMR spectroscopy.
19. 1 one – Dimensional SDS-PAGE
2 Two- Dimensional SDS-PAGE
What is SDS-PAGE? – SDS-PAGE a type of gel
electrophoresis.
What is the purpose of doing gel electrophoresis? –
It has been seen that by running a gel we are able to
identify more proteins from the sample.
An electric current is applied across the gel, causing
proteins will differentially migrate based on their
molecular mass.
20.
21. 2-D gel electrophoresis a method for the separation and
identification of proteins in a sample by displacement in 2
dimensions.
First step is to separate based on charge or isoelectric
point, called isoelectric focusing.
Then separate based on size (SDS-PAGE).
22.
23.
24. Mass spectrometry is used for protein identification.
It is useful to obtain structural information like peptide
mass sequence.
It is also useful in identifying type and location of protein
modification.
A mass spectrometer separates proteins according to their
mass-to-charge(m/z) ratio. The molecule is first ionized.
The process of ionization of proteins forces them to move
towards the analyzer because of the charges on ions.
MS can provide molecular weight and structural
information.
MS always work with positive ions.
25. While 2D- gel electrophoresis separates proteins, it
doesn’t identify them. MS is used to identify them which
separates charged particles or ions according to mass.
2 types of MS instruments
1. MALDI-TOF – matrix assisted laser desorption
ionization-time of flight. MALDI-is method of ionization
TOF- is a mass analyzer
2. ESI Tandem mass analyzer[ ESI-MS-MS] ESI-
process by which the ions are produced in the source of
the instrument Tandem mass analyzer- are able to perform
2 stage[multistage mass analysis]
26.
27. 1 source- produces gas-phase ions from the sample.
2 mass analyzer- resolves ions based on their m/z ration.
3 Detector- detects ions resolved by the mass analyzer.
28. • Protein fingerprinting , also called peptide mass
fingerprinting or peptide mapping.
• It is a technique for identification of proteins.
• Separated protein spots are obtained from the gel and then
identified using protein fingerprinting. The method is based
on the use of a proteolytic enzyme to digest the protein into
a number of smaller peptides.
• Unknown protein of interest cleaved into peptide by
protease.
• The most commonly used protease is trypsin, which cuts
protein at lysine and arginine positions.
• When the digestion in complete, a set of peptides are
produced of varying masses that are unique to that protein.
29.
30. Another strategy for large scale study of proteins, similar to
DNA microarray.
Procedure: A very small amount of different purified
proteins are placed on a glass slide in a pattern of rows and
columns.
Followed addition of various types of the probe molecules,
that are fluorescent dye labeled, to the array.
When the probe binds to the label it results in fluorescent
signal that can be ready by a laser scanner
Thus this technique can detect thousands of protein
interaction, can screen the ability of protein to bind other
proteins in complex, receptors, antibodies, lipid, enzymes,
hormones, specific DNA sequences or small molecules such
as new drugs.
33. • Most of the yeast two- hybrid systems utilize the
reconstitution of an active transcription factor to
assay for protein-protein to make in interactions.
• The Y2H system uses the trancription process to
make the prediction about protein interaction.
• The system requires that two yeast hybrids be
prepared called “bait-prey” system.
• The “bait” protein is fused to a transcrition factor
DNA binding domain.
• The other “prey” protein is fused to a transcription
factor activation domain.
34. • When expressed in a yeast cell containing the
appropriate reporter gene, interaction of the “bait”
with the “prey” brings the DNA binding domain and
the activation domain into close proximity, creating a
functional transcription factor.
• The ‘ bait prey’ nomenclature has applied to in vitro
method used to study protein analysis.
• In vitro method for protein interaction analysis are
often employed to confirm interaction indicated by
the Y2H method.
35.
36. Data analysis search engines
Sequest
MASCOT
X tandam
Peaks
Protein data bases
NCBS
Swiss port
37.
38.
39.
40. Protein sample identification/ confirmation.
Protein sample purity determination.
Detection of post-translational modifications
Detection of amino acids substitution.
Mass fingerprint identification of proteins.
Nutrition Research
To identify unknown protein of intrest.
Quantify protein and peptide.
Protein Biomarker.
41. 1. Proteomics is a composite study of a set of proteins .
2.The detailed protein studies will shed light on the role of
Protein modification in protein function.
3.The development of proteomics renders us with a
powerful tool to examine biochemical processes at the
molecular level and identify sets of proteins.
4. During plant life some times in adverse conditions it can
passes through biotic/abiotic stresses, at a time
proteomics can useful to identify sets of proteins.
