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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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. Cardiovascular Neuromuscular Islet cell Endothelial cell Muscle cell. - PowerPoint PPT Presentation

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Page 1: Sequencing of Mammalian Genomes Predicts 30,000 genes

Sequencing of Mammalian Genomes Predicts 30,000 genes

2001 2002

Page 2: Sequencing of Mammalian Genomes Predicts 30,000 genes

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

Page 3: Sequencing of Mammalian Genomes Predicts 30,000 genes

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.

Page 4: Sequencing of Mammalian Genomes Predicts 30,000 genes

Will Proteomics Provide a Stronger Means to Stratify Disease

Greater Complexity=Greater Specificity

YES

Page 5: Sequencing of Mammalian Genomes Predicts 30,000 genes

Protein Modifications

PhosphorylationGlycosolationUbiquitinationCleavageLipid etc.

Dictates Function and Intracellular Localization

Page 6: Sequencing of Mammalian Genomes Predicts 30,000 genes

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

Page 7: Sequencing of Mammalian Genomes Predicts 30,000 genes

How Gene and Protein Expression is Regulated or Modified from Transcription to

Post-translation

Page 8: Sequencing of Mammalian Genomes Predicts 30,000 genes

Synaptojanin 2

Page 9: Sequencing of Mammalian Genomes Predicts 30,000 genes

Biomarker ID vrs Protein ID

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

PROTEIN• INTERACTION• PRESENCE• ISOFORMS• MODIFICATIONS• FUNCTION

Page 10: Sequencing of Mammalian Genomes Predicts 30,000 genes

Using Proteomics to Identify Biomarkers of Disease

Page 11: Sequencing of Mammalian Genomes Predicts 30,000 genes

How do You ID Biomarkers

• 2D-PAGE MALDI-TOF MS

• ICAT MALDI-TOF MS• SELDI-TOF MS

Page 12: Sequencing of Mammalian Genomes Predicts 30,000 genes

Proteomic flow

1D and 2D gels

Proexpress Imager Progest ProMSPropicker

Q Star XL-MS/MS-TOF

Vision StationBiacore

Page 13: Sequencing of Mammalian Genomes Predicts 30,000 genes
Page 14: Sequencing of Mammalian Genomes Predicts 30,000 genes
Page 15: Sequencing of Mammalian Genomes Predicts 30,000 genes

ICAT(Isotope Coded

Affinity Tag)

Deuterium

Biotin Linker Thiol ReactiveGroup

Page 16: Sequencing of Mammalian Genomes Predicts 30,000 genes

ICAT Provides Relative Levels of

Expression Between Two

Proteins Which is

Reflected by the Ratio of

the Amount of Peptide

Page 17: Sequencing of Mammalian Genomes Predicts 30,000 genes

Surface Enhanced Laser Desorption Ionization Time of Flight (SELDI-TOF)

ProteinChip

Page 18: Sequencing of Mammalian Genomes Predicts 30,000 genes

Protein Chip

Page 19: Sequencing of Mammalian Genomes Predicts 30,000 genes

Different Protein Profiles from Different Chip Surfaces

Page 20: Sequencing of Mammalian Genomes Predicts 30,000 genes
Page 21: Sequencing of Mammalian Genomes Predicts 30,000 genes

Laser Capture

Microscopy Allows for the Dissection of Cells Directly

from Sectioned Tissues

Page 22: Sequencing of Mammalian Genomes Predicts 30,000 genes

LCM of Normal and Cancerous Prostate

Page 23: Sequencing of Mammalian Genomes Predicts 30,000 genes

Protein Profile from Prostrate Cancer Tissue

Procured by LCM

Page 24: Sequencing of Mammalian Genomes Predicts 30,000 genes

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