Proteomics 2

Question 1

What is proteomics?

What is the proteome?

Answer 1

Question 2

Why proteomics?

Answer 2

Question 3

What processes are involved in proteomics?

Answer 3

Proteomics uses knowledge of the structure of the proteins the proteins are made up of - AA of different weights and charges.

There is 2 steps involved: First 2D gele electrophoreis and then mass spectrometry

Question 4

Describe the seperation stage via gel electorphoresis of proteins

Answer 4

2D gel electrophoresis is used to carry out the seperation of proteins based on 2 characteristics: Isolectric point (charge) and molecular weight.

Seperation in the first dimension is by isolelectric point and in the second dimension is by molecular weight of proteins.

The proteins are run by electrophoreis on agrose gel and first sepeated by isolectric point (the pH to which the protein has an overall net charge of zero).

After seperation based on charfe the proteins are seperated based on mass. This is done by equilibriatn the first agrose gel with a negtaive surfacant called sodium dodecyl sulfate which coats all the proteins with a negative charge. This means all proteins have the same charge so seperation can occur due to differences in mass souly. This gel is put ontop of a poly acrylica gel and seperation occurs via mass.

These 2 properties are a very powerful means of seperation of complex mixtures of proteins.

After electophoresis the proteins are then stained with either:

• Silver
• Coomassie
• Fluorescent stains (SYPRO ruby)

It is important the dye is compatable with time of flight mass spec.

Question 5

What can 2D-page electrophoresis show us?

What is Nothern, southern and western bloting?

Answer 5

NOthern - DNA hybridisation (probing DNA)

Southern - looking at RNA

Western - looking at proteins

Question 6

After 2D gel electrophoresis has been carried out protein identification occurs vis generation of a peptide mass fingerprint using in flight mass spec

Describe this process

Answer 6

Variety of data bases can be compared against

Question 7

Validation of methods

Answer 7

Test against other patient samples to see if have specific marker we want

Question 8

Describe Laser capture microdisection (LCM)

Answer 8

• LCM is a straightforward method for obtaining cells for molecular analysis
• Direct microdissection of cells from tissue sections.
• Cells usually identified and selected on the basis of their morphology,
  
  • immunohistochemical phenotype
    
    • Tissues stained to allow for identification of different types of cell or tissue
  • or
  • genotype, after in situ hybridisation.

Only extracting specific cells intrested in

Question 9

Describe Reverse phase protein microarray (RDMA)

Answer 9

Question 10

Describe protein chips

Answer 10

AB chip —-> Test for antigens —–> Antigen-AB

Antigen chip —-> Test for AB present —> Antigen - AB

Protein chip —> Drug, protein, liposome, substrate —> -p-p, p-d, p-l, enzyme-substrate

Question 11

Describe the SELDI array technology

Answer 11

Question 12

Summary

Answer 12

• The study of expressed or synthesized proteins has greatly aided in the characterization of promising biologic therapies by providing information where genomics and previous analytical techniques were inadequate.
• Examples of recent proteomic techniques include mass spectrometry, which yields pertinent information on post-translational modifications, high-pressure liquid chromatography (HPLC), which allows for visual separation of proteins, and nuclear magnetic resonance (NMR) spectroscopy for structure determination.
• The advancement of these techniques continues to provide useful information to the field of protein science.
• Protein sequence, and subsequently protein sequence databases, are much more complex than DNA
• Prediction of protein structure is a complex problem at both the 2D and 3D levels
• Proteomics initiatives based on different technologies are making inroads into the study of protein structure and function on a global level

Question 13