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Front 1

What is proteomics?

Back 1

It is the large scale study of proteins, particularly their structures and functions.

Front 2

What is proteomics analogous to ?

Back 2

genomics - the study of an organisms collection of genes

Front 3

What is a genome?

Back 3

a relatively constant collection of genes

Front 4

What is the proteome of the organism?

Back 4

The entirety of proteins in existence in an organism throughout its life cycle

Front 5

What is the proteome of a cell type?

Back 5

The entirety of proteins found in a particular cell type under a particular type of stimulation

Front 6

Proteome differs from cell to cell and constantly changes through what?

Back 6

the biochemical interactions with the genome and the environment

Front 7

One organism has radically different protein expression in what?

Back 7

different parts of the body, at different stages of its life cycle, in different environmental conditions

Front 8

classical doctrine of gene functions

Back 8

1 gene leads to one protein which leads to one function

Front 9

Current View on Human gene function

Back 9

30,000 genes leads to 1,000,000 proteins which leads to multiple functions

Front 10

What are the nine branches of Proteomics?

Back 10

1. Protein Separation
2. Protein Identification
3. Protein Quantification
4. Protein Sequence Analysis
5. Structural Proteomics
6. Interaction Proteomics
7. Protein Modification
8. Cellular Proteomics
9. Experimental Bioinformatics

Front 11

All proteomic technologies rely on the ability to do what?

Back 11

separate a protein of interest from a complex mixture so that it can be further processed by other technologies

Front 12

An assay can be developed from what?

Back 12

any method that can be employed to distinguish the target protein from hundreds or thousands of others

Front 13

structural assays are based on what ability?

Back 13

the ability to specifically identify unique structural features. usually some form of immuno-assay (RIA, ELISA), but may involve other ligands.

Front 14

Structural assays can identify between what types of proteins?

Back 14

activated and unactivated

Front 15

What are a few examples of specific arrays?

Back 15

purification, fractionation, chromatography, analysis

Front 16

What is the most important step in protein purification?

Back 16

Need to develop a specific assay

Front 17

Functional assays are best suited for ________

Back 17

enzymes, but may also be extended to essential cofactors

Front 18

Functional assays can distinguish between what types of proteins?

Back 18

Denatured and fully functional

Front 19

What is protein purification?

Back 19

A systematic stepwise process of enrichment in target material. It exploids a variety of molecular characteristics that favor the selection of target protein

Front 20

The steps in protein purification are designed so that one is able to do what?

Back 20

1. remove large amounts of unwanted material early on.
2. remove decreasing levels of impurities later on

Front 21

What is fractionation?

Back 21

any technique leading to selective precipitation of desired protein or a larger portion of unwanted material.

Front 22

Fractionation most commonly uses what?

Back 22

SALT (ammonium sulfate)or any suitable solute:
1. salting in- the process of rendering a poorly soluble protein more soluble by adding the addition of salt. complements the ionic charges of the protein and allows it to dissolve.
2. salting out - the precipitation of a soluble protein by the addition of salt

Front 23

Can the same salt concentration be used for all proteins in fractionation?

Back 23

No each protein has its own ideal salt concentration and salting properties

Front 24

Describe the three steps in Chromatography?

Back 24

1. protein mixture is dissolved in a liquid (mobile phase)
2. this solution is passed across a solid (stationary phase)
3. proteins separate from each other based on how they distribute between these two phases

Front 25

What are four distinct types of chromatography?

Back 25

1. ion-exchange (anion or cation)
2. hydrophobic
3. affinity
4. gel filtration

Front 26

What is the purpose of protein analysis?

Back 26

quality control - to see the presence of contaminants

Front 27

What are two common structural assays?

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1. 1D PAGE (Polyacrylamide Gel Electrophoresis) - resolve by protein molecular size (smallest proteins move the farthest in the gel)
2. 2D PAGE- resolve through isoelectric properties

Front 28

How does ion exchange chromatography work?

Back 28

it separates proteins on the basis of their charge. anion-exchange resins, which consist of positively charged materials, bind reversibly with a protein's negatively charged groups. Cation-exchange resins bind positively charged groups. after proteins that do not bind to the resin are removed, the protein of interest is recovered by an appropriate change in solvent pH and/or salt conc.

Front 29

How does gel filtration chromatography work?

Back 29

the stationary phase is a gelatinous polymer with pore sizes selected by the experimenter to separate molecules according to their sizes. the sample is applied to the top of the column and is eluted with buffer. as elution proceeds, larger molecules travel faster through the gel than smaller molecules, whose progress is slowed b/c they can enter the pores. if fractions are collected the larger molecules appear in the earlier fractions and later fractions contain smaller molecules

Front 30

What are three ways a protein can be identified?

Back 30

1. sequencing through Edman degradation
2. mass spectrometry (particularly with peptide mass fingerprinting) - tryptic digest of protein followed by LC/MS and comparison of peptide elution profile and masses to available database information
3. antibody-based assay (unique to a particular structural epitope)

Front 31

How does mass spectrometry work?

Back 31

molecules are vaporized and then bombarded with a high energy electron beam causing them to fragment as cations. as the ionized fragments enter the spectrometer they pass through a strong magnetic field that separates them according to their mass-to-charge ration. each type of molecule is identified by the pattern of fragments that is generated

Front 32

What is protein quantification useful for?

Back 32

comparing the protein profiles in different cells - you can see which proteins are present

Front 33

Explain three ways protein quantification can be carried out

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1. Spectrophotometry - most effective for purified proteins
2. Gel-based methods, densitometry, specific fluorescent dyes
3. Microarry techniques - yield a profile of varying protein levels under a variety of conditions or from different cell-type sources

Front 34

What is structural proteomics?

Back 34

determination of protein structures in 3-D space

Front 35

What are two ways to determine a proteins 3-D structure in space?

Back 35

X-ray Crystallography
NMR Spectroscopy - looks at protein in solution

Front 36

Describe x-ray crystallography

Back 36

highly ordered crystalline specimens are exposed to an X-ray beam. as the X-rays hit the crystal, they are scattered by the atoms in the crystal. The diffraction pattern that results is recorded on a photographic plate and used to construct an electron density map. the 3D image is reconstructed mathematically

Front 37

What is the x-ray source in x-ray crystallography?

Back 37

rotating copper anode

Front 38

Much of the cells work is not done by individual proteins but is done by what?

Back 38

large macromolecular protein complexes

Front 39

List 7 qualitative/semi-quantitative protein:protein binding studies

Back 39

1. pull-down assays
2. coiummunoprecipitation
3. cross-linking
4. dialysis
5. affinity chromatography
6. ELISA
7. bia-core

Front 40

List 6 quantitative protein:protein binding studies

Back 40

1. light scattering
2. fluorophore perturbation
3. fluorescence anisotropy
4. fluorescence resonance energy transfer
5. fluorescence lifetime
6. microcalorimetry

Front 41

Describe the FRET (fluorescen resonance energy transfer)Approach

Back 41

A donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close proximity.When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence

Front 42

Describe surface plasmon resonance (Biacore)

Back 42

surface plasmon waves are excited at a metal/liquid interface. light is detected at and reflected from the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination angle and wavelength. biomolecular binding events cause changes in the refractive index at the surface layer which are detected as changes in the SPR signal.

Front 43

Protein Characterization Summary

Back 43

1. determine basic characterisitics : size, molecular weight, isoelectric point, native conditions (pH, salt, temp), and if enzyme know inhibitors effective
2. amino acid composition
3. digestion with selected proteases to obtain peptides
4. identify AAs that have been post-translationally modified
5. structural studies - X-ray crystallography, NMR, CD
6. interaction studies - equilibrium binding studies
7. functional studies - kinetics