Chromatography, Antibodies, and Proteomics

Question 1

Who was the term “proteomics” first presented by?

Answer 1

Marc Wilkens

Question 2

In 1994, what did proteomics describe?

Answer 2

The protein complement of genomics

Question 3

What did proteomics provide?

Answer 3

Large-scale analysis of proteins under a given set of conditions

Question 4

What is a large challenge of proteomics?

Answer 4

Dynamic nature of protein expression and range of concentration in cellular material, but it can also reveal critical mechanistic insights into cellular processes

Question 5

What are the application of proteomics?

Answer 5

• Protein profiling
• Biomarker discovery
• Cellular dynamics

Question 6

What is protein profiling?

Answer 6

Inventory of proteins at the levels of sub-organelles, cells, tissue, and organism

Question 7

What do biomarkers identify?

Answer 7

Diagnostic markers for diseases and therapeutic monitoring

Question 8

What does cellular dynamics do?

Answer 8

Quantify proteomic changes in response to various cellular environments and disease states

Question 9

What does comparative proteomics do?

Answer 9

Measures the expression of a set of proteins in two samples so they can be compared

Question 10

What are examples of comparative proteomic uses?

Answer 10

• 2D gel electrophoresis with DIGE technology
• LC-MS/MS using quantitative mass spectrometry

Question 11

What are different strategies for proteome purification and different protein separation prior to identification of mass spectrometry?

Answer 11

• Separation of individual proteins by 2D electrophoresis
• Multidimensional chromatography

Question 12

What are some problems of separating individual proteins by 2D electrophoresis?

Answer 12

• Acidic/basic proteins
• Membrane proteins
• Low abundant proteins

Question 13

What does multidimensional chromatography do?

Answer 13

Separate mixture of analytes into individual components (can be interfaced with electrospray-mass spectrometry systems)

Question 14

What is MudPIT?

Answer 14

Multidimensional protein identification technology

Question 15

What does two dimensional liquid chromatography do?

Answer 15

Separate crude protein mixture based on charge and hydrophobicity and couple to mass spectrometer for ID

Question 16

During the large-scale analysis of the yeast proteome, what was MudPit applied to?

Answer 16

The proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date
- 1,484 proteins detected and identified

Question 17

What did the categorization of 1,484 protein hits demonstrate?

Answer 17

The ability of this MudPIT to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors, protein kinases, and transmembrane proteins

Question 18

What is the overview of immunoprecipitation and interactome analysis?

Answer 18

• Stationary phase is resin functionalized with an antibody specific to the protein of interest
• Eluted using a variety of methods (pH, salt, competitive ligand, SDS loading dye, trypsin)
• Mass spectrometry is used to identify known and unknown proteins

Question 19

What can immunoprecipitation and interactome analysis be used for?

Answer 19

Rapid and unbiased method to discover novel functions of protein and isolate other proteins that interact with your protein of interest