Biotech Final - Lecture 7

Question 1

Outline the steps from lysing animal cells to analyzing (not identifying) a protein of interest.

Answer 1

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

Question 2

What are proteomics?

Answer 2

Systemic analysis of the proteins expressed in a cell under a given set of conditions.

Question 3

The proteome is only analyzed once when?

Answer 3

The proteome forms a database.

Question 4

The proteome is analyzed multiple times when?

Answer 4

When we wish to detect dynamic changes in the proteome following external or internal perturbations. Thus, proteomics can be a biological/clinical assay.

Question 5

A proteome is defined by what?

Answer 5

The state of the organism, tissue or cell that produces it.

Question 6

What are the different applications of proteomics?

Answer 6

1) Comparative expression analysis
2) Characterization of biological processes
3) Biomarkers
4) Drug analysis

Question 7

What is comparative expression analysis?

Answer 7

Compare disease proteome vs normal proteome or response to external stimuli

Question 8

What does characterizing biological processes by proteome analysis entail?

Answer 8

Characterize sub-proteomes such as protein complexes, cellular networks, etc…

Question 9

How can proteomics serve as biomarkers?

Answer 9

Finding certain proteins overexpressed may serve as a diagnostic for disease.

Question 10

How can proteomics be applied to drug analysis?

Answer 10

Evaluate toxicity & other biological or pharmaceutical parameters associated with drug treatment.

Question 11

What is comparative proteomics?

Answer 11

Measure the expression of a set of proteins in two samples and compare them.

Question 12

What are the different comparative proteomics methods?

Answer 12

1) 2D gel electrophoresis

2) DIGE

Question 13

What is 2D gel electrophoresis?

Answer 13

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.

Question 14

What is DIGE?

Answer 14

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.

Question 15

DIGE overcomes what major constraint of 2D-Gel electrophoresis?

Answer 15

Overcomes the major constraint of non-biological variation due to between-gel variation.

Question 16

What are the different strategies for proteome purification and protein separation prior to identification by MS?

Answer 16

1) 2D-gel electrophoresis

2) Multidimensional chromatography (ex: MudPIT)

Question 17

What are some of the issues with separation of individual proteins by 2D-gel electrophoresis, in what concerns proteome purification?

Answer 17

Problems with

1) acidic/basic proteins (poorly separated)
2) membrane proteins (insoluble proteins)
3) low abundant proteins

Question 18

What is MudPIT? (Very broadly)

Answer 18

Two dimensional liquid chromatography. Separate crude protein mixture based on both charge and hydrophobicity. This is coupled to mass spectrometer for identification.

Question 19

What are the general steps for MudPIT?

Answer 19

1) Load acidified digest
2) Equilibration
3) Salt step
4) Wash
5) RP gradient
6) Repeat step 2 onward

Question 20

MudPIT has the ability to detect multiple proteins which include?

Answer 20

Low abundance proteins, TFs, protein kinases, transmembrane proteins

Question 21

Describe how MudPIT works to identify proteins.

Answer 21

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

Question 22

What are the pitfalls of MudPIT?

Answer 22

Cation exchange resin can mix with RP resin and needle can get clogged.

Question 23

How were the issues with MudPIT fixed?

Answer 23

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.

Question 24

What is the automated protein identification strategy?

Answer 24

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.

Question 25

What is TAP?

Answer 25

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.

Question 26

What are the advantages of TAP? What are the disadvantages?

Answer 26

Eliminates non-specific binding issues.

Not good for low affinity binding complexes.