sequencing of mammalian genomes predicts 30,000 genes

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

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

Greater Complexity=Greater Specificity

YES

Protein Modifications

PhosphorylationGlycosolationUbiquitinationCleavageLipid 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

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

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 Progest ProMSPropicker

Q Star XL-MS/MS-TOF

Vision StationBiacore

ICAT(Isotope Coded

Affinity Tag)

Deuterium

Biotin Linker Thiol ReactiveGroup

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)

ProteinChip

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