Sequencing of Mammalian Genomes Predicts 30,000 genes

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Sequencing of Mammalian Genomes Predicts 30,000
genes
2001
2002
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What is the Proteome

• All the proteins expressed in a particular cell
  or tissue.
• By definition this will vary by tissue and cell
  type.
• Cardiovascular
• Neuromuscular
• Islet cell
• Endothelial cell
• Muscle cell

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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.

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Will Proteomics Provide a Stronger Means to
Stratify Disease
YES
Greater ComplexityGreater Specificity
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Protein Modifications

• Phosphorylation
• Glycosolation
• Ubiquitination
• Cleavage
• Lipid etc.

Dictates Function and Intracellular Localization
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30,000 Genes How Many Proteins?
30,000 genes
4-6 splice variants
120,000 possible mRNAS
gt200 Post-translational Modifications
24,000,000 Possible Protein Isoforms
2,000,000 Predicted Proteins
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How Gene and Protein Expression is Regulated or
Modified from Transcription to Post-translation
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Synaptojanin 2
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Biomarker ID vrs Protein ID

• BIOMARKER
• CHANGE /-
• IDENTITY ?
• SPECIFIC
• EASE
• BODY FLUID

• PROTEIN
• INTERACTION
• PRESENCE
• ISOFORMS
• MODIFICATIONS
• FUNCTION

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Using Proteomics to Identify Biomarkers of Disease
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How do You ID Biomarkers

• 2D-PAGE ? MALDI-TOF MS
• ICAT ? MALDI-TOF MS
• SELDI-TOF MS

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Proteomic flow
1D and 2D gels
Proexpress Imager
Progest
ProMS
Propicker
Q Star XL -MS/MS-TOF
Biacore
Vision Station
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Centre for Proteomic and Genomic Studies
vision workstation
agilent lab-on-chip
Q-Star-XL MS/MA-TOF
Protein Isoform Characterization Lab
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ICAT(Isotope Coded Affinity Tag)
Deuterium
Thiol Reactive Group
Biotin
Linker
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ICAT Provides Relative Levels of Expression
Between Two Proteins Which is Reflected by the
Ratio of the Amount of Peptide
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Surface Enhanced Laser Desorption Ionization Time
of Flight (SELDI-TOF)
Protein Chip
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Protein Chip
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Different Protein Profiles from Different Chip
Surfaces
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Laser Capture Microscopy Allows for the
Dissection of Cells Directly from Sectioned
Tissues
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LCM of Normal and Cancerous Prostate
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Protein Profile from Prostrate Cancer Tissue
Procured by LCM
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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