Transcript Proetomics and Signaling

Sequencing of Mammalian
Genomes Predicts 30,000 genes
2001
2002
What is the Proteome
• All the proteins expressed in a particular
cell or tissue.
• By definition this will vary by tissue and
cell type.
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Cardiovascular
Neuromuscular
Islet cell
Endothelial cell
Muscle cell
Why Study Proteomics?
• Gives a better understanding of the
function of gene products.
• Allow for the design of rational drug
therapies.
• Provide new and specific markers of
disease.
Will Proteomics Provide a Stronger
Means to Stratify Disease
YES
Greater Complexity=Greater Specificity
Protein Modifications
Phosphorylation
Glycosolation
Ubiquitination
Cleavage
Lipid etc.
Dictates Function and
Intracellular Localization
30,000 Genes How Many Proteins?
30,000 genes
4-6 splice variants
120,000 possible mRNAS
>200 Post-translational Modifications
24,000,000 Possible Protein Isoforms
2,000,000 Predicted Proteins
How Gene and Protein Expression is
Regulated or Modified from Transcription to
Post-translation
Synaptojanin 2
Biomarker ID vrs Protein ID
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BIOMARKER
CHANGE +/IDENTITY ?
SPECIFIC
EASE
BODY FLUID
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PROTEIN
INTERACTION
PRESENCE
ISOFORMS
MODIFICATIONS
FUNCTION
Using Proteomics to Identify
Biomarkers of Disease
How do You ID Biomarkers
• 2D-PAGE  MALDI-TOF
MS
• ICAT  MALDI-TOF MS
• SELDI-TOF MS
Proteomic flow
1D and
2D gels
Proexpress Imager
Propicker
Progest
Q Star XL
-MS/MS-TOF
Vision Station
Biacore
ProMS
ICAT
(Isotope
Coded
Affinity Tag)
Deuterium
Biotin
Linker
Thiol Reactive
Group
ICAT Provides
Relative
Levels of
Expression
Between Two
Proteins
Which is
Reflected by
the Ratio of
the Amount of
Peptide
Surface Enhanced Laser Desorption Ionization
Time of Flight (SELDI-TOF)
Protein
Chip
Protein Chip
Different Protein Profiles from
Different Chip Surfaces
Laser
Capture
Microscopy
Allows for the
Dissection of
Cells Directly
from
Sectioned
Tissues
LCM of Normal and Cancerous
Prostate
Protein
Profile from
Prostrate
Cancer
Tissue
Procured by
LCM
Do we Need all Three Approaches?
• 2D-PAGE  MALDI-TOF MS
– Greatest resolving power
– Large data base
– Labor and time consuming
• ICAT  MALDI-TOF MS
– Mostly abundant proteins
– Needs cysteine residue
• SELDI-TOF MS
– Resolves from 8-50 kDa
– Small amounts of sample