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Proteomics ( Role in Reproduction Studies)

Proteomics ( Role in Reproduction Studies). Jayanti Tokas 1 , Puneet Tokas 2 , Shailini Jain 3 and Hariom Yadav 3 1 Department of Biotechnology, JMIT, Radaur, Haryana, India 2 KITM, Kurukshetra, Haryana, India 3 NIDDK, National Institute of Health, Bethesda, MD 20892, USA

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Proteomics ( Role in Reproduction Studies)

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  1. Proteomics (Role in Reproduction Studies) Jayanti Tokas1, Puneet Tokas2, Shailini Jain3 and Hariom Yadav3 1Department of Biotechnology, JMIT, Radaur, Haryana, India 2KITM, Kurukshetra, Haryana, India 3NIDDK, National Institute of Health, Bethesda, MD 20892, USA Email: yadavhariom@gmail.com

  2. PROTEOMICS • The term ‘Proteome’ coined in 1994 • Complete set of proteins exported and modified following expression, by entire genome in lifetime of a cell • Proteomics is usually carried out to study the complement of protein expressed by a cell at any one time or at a particular stage

  3. Why Proteomics? • Genome: blueprint of the cell. • Protein encoding genes: 60,000 • 6-8 proteins/gene. • Thousands proteins due to PTM • and splice variants.

  4. Proteomics tools

  5. 2 D electrophoresis • 1. Proteins migrate under electric field on the basis of • conformation • size • electric charge 2 It uses: • Iso electro focussing in one dimension (charge) • SDS-PAGE in second dimension (mol.wt) • Each spot represent one to few proteins depending on the complexity of sample used.

  6. Mass Spectrometry • Mass measurement of ions derived from molecules • Capable of forming, separating & detecting ions on the basis of m/z values. Sample introduction Ion formation Ion separation Data handling Data output

  7. MALDI- Matrix Associated Laser Desorption Ionization (Yates et al.1998)

  8. ESI: Electron Spray Ionization

  9. Mass can be calculated by: P1 = (M + z1)/ z1 P2 = (M+ z1-1)/ z1-1 Figeys et al.2001

  10. Mass analyzers: Quadrupoles • Consist of 4 parallel metal rods • Two opposite rods have +ve potential and other two have –ve potential (AC & DC) • Applied voltage influence trajectory of ions traveling down flight path centered bw two rods • For given voltage, only certain m/z ions will reach detector

  11. Tandem MS/MS • Mass selection of precursor ion in first stage • Fragmentation of ions by collision induced dissociation (CID) • Ions produced are named as: • NH2 terminal COOH terminal • C-C: a x • C-N b y • N-C c z • Ions produced: 2nd analysis. • Sequencing of proteins.

  12. Gel free approach Gel approach is not suitable for Hydrophobic and highly charged proteins as they disturb the movement in gel ICAT: 14% of eukaryotic Proteins lack Cysteine not compatible with that. (Aebersold et al. 1999)

  13. MuDPIT: Multi Dimensional Protein Identification Technique • Whole protein mixture is digested with trypsin • Stepwise separation by CEX (Cation exchange Column in one Dimension and RP-HPLC in other • Series of column with MS/MS • Protein identification

  14. Protein – Protein Interactions • Protein Microarrays: • Capture molecules used: • Antigens • Aptamers • Substrates • Ligands • Carbohydrates etc. Microarrays and SELDI: The chips so formed can be analyzed directly by MALDI-MS called Surface Enhanced Lazer Desorption Ionization

  15. Two Hybrid Assay Uetz et al, 2000

  16. Overview of Oocyte Maturation and Implantation

  17. Protein patterns during in vitro maturation • Methodology: oocyte collection and culture, 2DE, gel digestion with trypsin, MALDI-TOF MS, LC-ESI, NCBI database search • Proteins abundantly found: • ZP sperm binding proteins: sulfated and highly O-glycosylated. (50- 70 Kda) • Peroxiredoxins: Thioredoxin dep. peroxide reductase localized in mt and cytoplasm • Might participate in signaling cascade • Spermine synthase: spermine from spermidine • Prevent from oxidative damage. • Protection from chromatin damage. • Molecular chaperons: HSP 60, Calreticulin, GRP 78. • Correct folding of protein. • Glycolytic enzymes:Enolase, TPI, 2-oxoglutarate Dhase, Galactokinase:

  18. Ub terminal hydrolase isozyme 1: involved in ubiquitination of proteins • Protein increased at MI and MII stage compared to GV stage: • Antiquitin:plant homologue protein responsive to turgor pressure • Evolutionary conserved protein • Share homology with ADH family • Found in outer outer hair cells in human ear for maintaining turgor pressure • Protective role against toxic effects of peroxide aldehydes produced by lipid peroxidation • Can act as biomarker for oocyte matutarion Ellederova et al, 2004

  19. THANK YOU

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