Proteomics (Role in Reproduction Studies)

Jayanti Tokas1, Puneet Tokas2, Shailini Jain3 and Hariom Yadav3

1Department of Biotechnology, JMIT, Radaur, Haryana, India2KITM, Kurukshetra, Haryana, India

3NIDDK, National Institute of Health, Bethesda, MD 20892, USAEmail: yadavhariom@gmail.com

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

Why Proteomics?

• Genome: blueprint of the cell.

• Protein encoding genes: 60,000

• 6-8 proteins/gene.

• Thousands proteins due to PTM and splice variants.

Proteomics tools

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.

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

MALDI- Matrix Associated Laser Desorption Ionization

(Yates et al.1998)

ESI: Electron Spray Ionization

Mass can be calculated by: P1 = (M + z1)/ z1

P2 = (M+ z1-1)/ z1-1 Figeys et al.2001

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

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 yN-C c z •Ions produced: 2nd analysis.•Sequencing of proteins.

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)

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

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

Two Hybrid Assay

Uetz et al, 2000

Overview of Oocyte Maturation and Implantation

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:

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