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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: [email protected]
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
y
N-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. (5070 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