proteomics

proteomics introduction
types of proteomics
objectives
protein structure
procedure of proteomics
proteomics techniques

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2. Assignment Presentation On PROTEOMICS Submitted by : Desai vruddhi k. M.Sc. (Agri.) GPB S.D.Agriculture University, Sardarkrushinagar PRINCIPLES OF BIOTECHNOLOGY (MBB 501)

3. PROTEOMICS

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. PROTEOMICS

5. GENOMICS TRANSCRIPTOMICS PROTEOMICS

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 . INTRODUCTION

7. 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. WHY PROTEOMICS ???

8. 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. Functional proteomics :- It refers to the use of proteomics techniques to analyze the characteristics of molecular protein-networks involved in a living cell. Expression proteomics :- It refers to the quantitative study of protein expression between sample differing by some variable. TYPES OF PROTEOMICS

9. Protein/peptide separation. Identification and characterization of resolved proteins by MS. Data analysis and applications. PROTEOMICS OBJECTIVES

10. 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. PROTEIN STRUCTURE

11. 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. conti …

12. Separation of proteins One dimentional electrophoressis 2 D electrophossis (modern) Multi-dimensional HPLC (modern) Analysis of proteins Mass Spectrometry (modern) Database utilization Procedure of proteomics

13. Sample collection, handling and storage. Separation of individual proteins by 2-D electrophoresis. Protein characterization. Identification by mass spectrometry or other methods. Storage, manipulation, and comparison of the data using bioinformatics . Five steps of proteome analysis

14. 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 PROTEOMICS TECHNIQUES

15. Techniques of Structural Proteomics The large-scale analysis of protein Protein structural comparisons can help to identify the function of newly discovered genes X-ray crystallography, NMR spectroscopy.

16. 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 . Protein separations Techniques

17. 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). 2-D gel electrophoresis

18. HPLC

19. 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. Mass Spectrometry

20. 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] Mass Spectrometry

21. 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. Components of a mass spectrometer

22. 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. Protein Fingerprinting

23. Protein Microarrays 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.

24. Protein-protein interaction Protein-protein interactions occur when two or more proteins bind together.

25. YEAST TWO-HYBRID SYSTEM FOR Protein-protein interaction 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.

26. 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.

27. Data analysis search engines Sequest MASCOT X tandam Peaks Protein data bases NCBS Swiss port DATA BASES

28. 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. Application of proteomics

29. 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. Conclusion