Chromatography, Antibodies, and Proteomics Flashcards
Who was the term “proteomics” first presented by?
Marc Wilkens
In 1994, what did proteomics describe?
The protein complement of genomics
What did proteomics provide?
Large-scale analysis of proteins under a given set of conditions
What is a large challenge of proteomics?
Dynamic nature of protein expression and range of concentration in cellular material, but it can also reveal critical mechanistic insights into cellular processes
What are the application of proteomics?
- Protein profiling
- Biomarker discovery
- Cellular dynamics
What is protein profiling?
Inventory of proteins at the levels of sub-organelles, cells, tissue, and organism
What do biomarkers identify?
Diagnostic markers for diseases and therapeutic monitoring
What does cellular dynamics do?
Quantify proteomic changes in response to various cellular environments and disease states
What does comparative proteomics do?
Measures the expression of a set of proteins in two samples so they can be compared
What are examples of comparative proteomic uses?
- 2D gel electrophoresis with DIGE technology
- LC-MS/MS using quantitative mass spectrometry
What are different strategies for proteome purification and different protein separation prior to identification of mass spectrometry?
- Separation of individual proteins by 2D electrophoresis
- Multidimensional chromatography
What are some problems of separating individual proteins by 2D electrophoresis?
- Acidic/basic proteins
- Membrane proteins
- Low abundant proteins
What does multidimensional chromatography do?
Separate mixture of analytes into individual components (can be interfaced with electrospray-mass spectrometry systems)
What is MudPIT?
Multidimensional protein identification technology
What does two dimensional liquid chromatography do?
Separate crude protein mixture based on charge and hydrophobicity and couple to mass spectrometer for ID
During the large-scale analysis of the yeast proteome, what was MudPit applied to?
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
What did the categorization of 1,484 protein hits demonstrate?
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
What is the overview of immunoprecipitation and interactome analysis?
- 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
What can immunoprecipitation and interactome analysis be used for?
Rapid and unbiased method to discover novel functions of protein and isolate other proteins that interact with your protein of interest
