Biotech Final - Lecture 7 Flashcards
Outline the steps from lysing animal cells to analyzing (not identifying) a protein of interest.
1) Lyse animal cells
2) Add protein A-agarose beads
3) Add antibody to beads, allow for binding
4) Add protein from lysed cell
5) Wash
6) Analyze by SDS, western or 2D gel
What are proteomics?
Systemic analysis of the proteins expressed in a cell under a given set of conditions.
The proteome is only analyzed once when?
The proteome forms a database.
The proteome is analyzed multiple times when?
When we wish to detect dynamic changes in the proteome following external or internal perturbations. Thus, proteomics can be a biological/clinical assay.
A proteome is defined by what?
The state of the organism, tissue or cell that produces it.
What are the different applications of proteomics?
1) Comparative expression analysis
2) Characterization of biological processes
3) Biomarkers
4) Drug analysis
What is comparative expression analysis?
Compare disease proteome vs normal proteome or response to external stimuli
What does characterizing biological processes by proteome analysis entail?
Characterize sub-proteomes such as protein complexes, cellular networks, etc…
How can proteomics serve as biomarkers?
Finding certain proteins overexpressed may serve as a diagnostic for disease.
How can proteomics be applied to drug analysis?
Evaluate toxicity & other biological or pharmaceutical parameters associated with drug treatment.
What is comparative proteomics?
Measure the expression of a set of proteins in two samples and compare them.
What are the different comparative proteomics methods?
1) 2D gel electrophoresis
2) DIGE
What is 2D gel electrophoresis?
Combines IEF and SDS-page. Use strip with pH gradient and apply voltage gradient, proteins travel through strip until they reach their pI. Then, take strip and glue onto SDS-PAGE gel.
What is DIGE?
Differential In-Gel Electrophoresis. Protein samples are pre-labeled with fluorescent dye, both with different max absorbances. These are then combined and run on the same IEF gel and then SDS-PAGE gel. This is followed by image analysis.
DIGE overcomes what major constraint of 2D-Gel electrophoresis?
Overcomes the major constraint of non-biological variation due to between-gel variation.
What are the different strategies for proteome purification and protein separation prior to identification by MS?
1) 2D-gel electrophoresis
2) Multidimensional chromatography (ex: MudPIT)
What are some of the issues with separation of individual proteins by 2D-gel electrophoresis, in what concerns proteome purification?
Problems with
1) acidic/basic proteins (poorly separated)
2) membrane proteins (insoluble proteins)
3) low abundant proteins
What is MudPIT? (Very broadly)
Two dimensional liquid chromatography. Separate crude protein mixture based on both charge and hydrophobicity. This is coupled to mass spectrometer for identification.
What are the general steps for MudPIT?
1) Load acidified digest
2) Equilibration
3) Salt step
4) Wash
5) RP gradient
6) Repeat step 2 onward
MudPIT has the ability to detect multiple proteins which include?
Low abundance proteins, TFs, protein kinases, transmembrane proteins
Describe how MudPIT works to identify proteins.
1) Take total cell lysate and trypsinize. (obtain crude lysate)
2) Apply to SCN (starling cation exchange)
2. a) elute using increasing salt concentration (do different salt bumps)
3) The eluted proteins bind to RP column (hydrophobic proteins bind stronger to this)
3. a) Elute using organic solvent (compatible for ESI into MS)
4. MS-TOF linear
5. MS-MS
6. Identify protein
What are the pitfalls of MudPIT?
Cation exchange resin can mix with RP resin and needle can get clogged.
How were the issues with MudPIT fixed?
2nd generation 2D systems.
1st generation is SCX
2nd generation is RP into MS.
There are alternating cycles that allow for the chromatography apparatuses to work simultaneously. No mixing of resins.
What is the automated protein identification strategy?
Lyse cells to obtain protein mixture Trypsinize to get cleaved peptides Separate by 1D, 2D or 3D separation MS-TOF linear MS-MS Compare observed with computer based in-silico Identify proteins.
What is TAP?
Two step affinity chromatography system.
Fusion protein with TAP tag. Tap tag has spacer-CBP-TEV site-protein A.
First affinity chromatography with IgG beads, binds to protein A. Wash then elute using TEV protease. Bring eluted fraction to second affinity chromatography column, calmodulin beads. Wash then elute. Determine binding partners.
What are the advantages of TAP? What are the disadvantages?
Eliminates non-specific binding issues.
Not good for low affinity binding complexes.
