Notes 20100504 Catherine Defense

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Comparative Proteomics

Catherine Wei
Tag: Chinese Hamster Ovarian Cells, Intrinsic Apoptosis Pathway, Proteome; no artificial induction
Intrinsic pathway to apoptosis: Mitochondrial membrane potential
Apoptosome a complex that activates Caspase 9 -- leads to executioner caspases etc.,
Intrinsic and extrinsic pathways linked at mitochondrion step
Exponential, stationary and death phases
Day 2.5 -- exponential phase (healthy)
Apoptosis starts at day 4.5 (the tail of exponential phase -- early stationary phase, apoptosis starts)
Accumulation of dead cells occurs at day 5.5 -- plasmalemmae start to leak
Day 8.5 -- death phase
Additional proteomic study guided by the above observed time points
Flow cytometry cell subtypes -- compare levels of caspase 8, caspase 9
12 DIGE gels result from comparison of three pairs of time points
Three heatshock proteins were down regulated (involved with cell survival)
Cleavage of cytoskeletal proteins
induce ER chaperones (=> Unfolded Protein Response (UPR) onset)
upregulation of six glycolysis proteins (enables completion of apoptosis in low energy environment)

Monoclonal antibody production
DIGE: Differential in gel electrophoresis
MS: Mass spectrometry (protein sequence determined, used to search database)
Flow cytometry
Propidium Iodide (PI) - Membrane permeability, viability stain
Protein major functions (in this study): Cytoskeleton, Chaperone, Metabolism
Cytoskeleton: Vimetin, α-tubulin, β-tubulin, actin(β/γ)
Endoplasmic reticulum chaperone systems -- facilitates protein folding; could have been triggered by ER stresses.