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Artificial Antibodies
- Used to make analytical arrays for protein capture
- Take antibodies and identify the variable portions
- let the immune system mature the T cells and allow the antibodies to undergo recombination events to rearrange variable parts
- can have more variety if you add mutations
- make a library of your antibodies and screen to find ones that bind to proteins
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Nanoparticles
- designed to sample across the proteome
- A sheer nanoparticle gathers a layer of proteins on its surface
- The binding of the proteins to the nanoparticles is based on the design of nanoparticle to be specific to targeted proteins
- Get information about what protein is in your sample and mix with antibodies to find the antibodies that interact with proteins
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Quantum dot tagged microbeads
- Small dots have specific flourescent properties
- combining the different colors allows you to have a large variety of quantum dots
- need to manage the dots to ensure the proteins don't denature
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Reverse phase protein arrays
- allows interrogation of a mixture of protein instead of a single purified protein
- quantifies proteins and protein transcriptional modifications in cellular lysases and body fluids
- suitable for biomarker discovery, protein pathway profiling, drug mode of action analysis
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What ways can we verify structural proteomics?
Crystal production (Xray crystallography)
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Types of Protein arrays:
- Functional arrays
- Analytical arrays
- Reverse phase protein arrays
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Describe the classical workflow to make a protein array
- purify protein
- express clone in your culture
- array the protein
- apply to an array application
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Describe in-situ protein arraying
- Express constructs generated by PCR
- Array the DNA
- In-situ synthesis of proteins
- apply to an array application
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What are the four ways you can attach proteins to an array?
- Diffusion: proteins suspended in random orientations, but active
- Adsorption: some proteins active
- Covalent attachment: some proteins active
- Affinity: orientation is controlled, and proteins are active
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What are the methods to read/visualize protein arrays?
- Fluorescence
- Surface enhanced laser desorption (SELDI)
- surface plasma resonance
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Describe how we might make a genome wide antibody array
- (we are not quite there yet)
- create artificial antibodies that can capture a wide variety of proteins
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2D gel electrophoresis (purpose)
- separates proteins by isoelectric point and by mass
- Can separate 500-2000 proteins and detect post-transnational modifications
- Has issues separating very acidic/basic proteins or membrane bound proteins
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2D gel electrophoresis (brief description)
- First dimension: run in a capillary tube to separate proteins by pH to their isoelectric points
- second dimension: run on a constant voltage across a gel to seperate proteins by size
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Describe the purpose of fractionation
- identify all proteins in a sample more thoroughly
- might fraction-ate by organelle type, antibodies, or biochemical properties of the protein
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How do we detect proteins in 2D electrophoresis?
- dyes help visualize proteins
- different dyes have different sensitives and dynamic range
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How do we recognize different proteins?
- antigen-antibody recognition
- N-terminal sequencing
- mass spectrometry!
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Types of Mass spec we discussed
- MALDI (and MALDI-TOF)
- Ion trap
- Tandem MS/MS
- Liquid Chromatography Tandem MS (LC/MS/MS)
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MALDI
- Matrix assisted Laser Desorption and Ionization
- fixed onto a matrix
- Shot with a laser to put into gas phase without decomposing the protein (ionize), and the gas is analyzed
- separated with a magnetic field
- proteins are identified based on their mass charge ratio
- MALDI-TOF has the addition of finding protein identity based on how long it takes to reach the detector
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Ion trap
- Form of mass spec, often paired with electrospray
- captures protein information on the basis of mass-to-charge ratio
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Tandem MS/MS
- sequence the readouts b/c the proteins could not be identified the first time run through a mass spec (might have too many post-transnational modifications)
- one peptide at a time, fractionate to further break apart proteins
- differences in pairs of proteins on the readout will depend on the mass of the amino acid that was lost in fractionation
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Liquid Chromatography Tandem MS (LC/MS/MS)
- First separates proteins by polarity
- mass charge separation comes after ionization
- can detect low abundant proteins, integral membrane proteins, proteins with extreme pIs
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Caveats of 2D gel electrophoresis
- does not work well with very large or very small proteins
- low-abundance proteins difficult to detect
- membrane-bound proteins cannot be detected
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ICAT
- isotope coding affinity tags quantification
- in situ uses stable isotopes to label proteins
- measure the abundance of proteins under different experimental conditions
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