Ch. 15 - Proteomics: The Global Analysis of Proteins Flashcards
Proteomics
The global analysis of proteins. Involves surveying the global protein composition of a cell or an organism. Used to study:
- Expression levels of genes
- Macromolecular interactions
- Post-translational modifications
- Signaling netwokrs
- Metabolic pathways
- Protein functions
- And more…
Proteomics: Study of all potential proteins a cell or organism can make, which is more proteins than genes. More than 100 000 proteins in the human, while 20 000 genes, more proteins than genes, due to alternative splicing, 5-15 different proteins from one gene, some more
Functional genomics
The study of how genes and intergenic regions of the genome contribute to different biological processes. The goal of functional genomics is to determine how the individual components of a biological system work together to produce a particular phenotype.
The study of the entire genome and its function. Includes proteomics, transcriptomics, metabolomics among others.
Functional genomics and proteomics:
1. Gene with exons
2. Transcript with exons
3.Alternative splicing, mRNA with a variety of exons
4. Gene products, polypeptides of different exons, different structure and functions
PTM, functional groups added, activation …
Proteome
Proteome: The total set of proteins encoded by a genome or the total protein complement of an organism.
Translatome: The total set of proteins that have actually been translated and are present in a cell under any particular set of conditions.
Protein characterization
Proteins differ from one another much more in both structure and function than nucleic acid molecules as they have 20 monomers to alter between instead of only 4.
The structure of a protein confers its function, and can be characterized by: Primary structure: - Identification, sequence validation - Characterization of splice variants Secondary structure: - Disulfide bonds - Protein folding domains Tertiary structure: - Determination of 3D structure - Protein structure dynamics Quaternary structure: - Kinetics and binding sites - Protein-protein interactions
Additionally, proteins have different isoelectric points (pI, the pH at which the protein has a net charge of zero), different charge, features, and sizes.
Quantification and Separation of proteins
Protein can be isolated by lysing the cell containing the protein, and then isolated. Quantification of proteins can be done through a variety of colorimetric assays. Two of the most common ones, Buiret reagent and Bradford assay, rely on copper chemistry and a protein-binding dye, respectively. In all these colorimetry methods, the amount of color produced is proportional to the amount of protein in the sample. The concentration of proteins can be determined by comparing the assay value to a set of known protein concentrations.
Proteins can be separated based on different features, for example size or charge. This can be done by column chromatography or gel electrophoresis. In column chromatography the proteins are separated due to their different affinity to the column based on size, charge, ions, or pH.
When proteins are separated by using gel electrophoresis, they are first denatured and coated with negatively-charged detergent molecules, such as sodium dodecyl sulfate (SDS). This detergent gives the protein a negative charge proportional to its length/size, as it is evenly distributed around the protein.
After denaturation, proteins can be separated by size by gel electrophoresis. As proteins are generally smaller than DNA and RNA, they are separated by polyacrylamide gel electrophoresis (PAGE). After running, the gel is stained with dye to visualize the protein bands.
2D Polyacrylamide gel electrophoresis (PAGE)
Separation of large numbers of proteins is normally done by 2D PAGE. The proteins are separated by original charge in the first dimension and then by size in the second dimension. After staining, the result of 2D PAGE is a square with small scattered dots representing individual proteins. Large 2D gels with high resolution have been developed that allow separation of over 10 000 spots and can be used to analyze the proteome of higher organisms.
In the first dimension, isoelectric focusing, which is a technique for separating proteins according to their charge by means of a electrophoresis through a pH gradient, is used to separate the native proteins based on their original charge. A pH gradient is set up along cylindrical gel and proteins migrate until they find a position where their native charges are neutralized - the isoelectric point (pI).
In the second dimension, standard SDS PAGE is used to separate the denatured proteins based on their molecular mass/size.
Protein identification methods
Proteins can be identified by mass spectrometry and several other methods:
- Purification of protein complex: proteins are extracted or tagged, multiple proteins can be bound/interacting in a complex
- Separation of protein components: different proteins have different features, gel electrophoresis or chromatography
- Identification of protein components: mass spectrometry, sequencing and data base searching
- Functional analysis: kinetics, function, interactions
Proteins can be digested into peptide fragments by enzymes trypsin, which cleaves at the carboxyl end of Lysine or Arginine. The masses of the peptide fragments can be determined by mass spectrometry (MS). The generated peptide fingerprint (characteristic masses) can be used to identify the protein. However, this requires either a standard for comparison or genomic data to search for a match. Peptides can be further fragmented (MS/MS) to give data from which the amino acid sequence can be calculated directly.
Protein isolation: Chromatography
Chromatography: General term for techniques that separate mixtures of components by using a mobile phase (liquid or gas) to carry the mixture over a stationary phase (solid or liquid). The proteins are eluted into multiple fractions, and the concentration of proteins in each of them can be monitored by UV light to create a chromatogram. Each peak on the chromatogram represents a fraction with pure protein.
Liquid chromatography of proteins: mixtures of dissolves proteins (mobile phase) are separated into fractions using columns packed with variations of solid materials.
Ion exchange chromatography (IEC): Separates a mixture of proteins based on their native charge. Affinity to the stationary phase (positively or negatively charged) determines the order in which the protein ions pass through the column, and thereby separation.
Hydrophobic Interaction Columns (HIC): Column with a matrix that binds to hydrophobic proteins. Other proteins pass through and are washed out.
High-Pressure Liquid Chromatography (HPLC): Separates a mixture of proteins through a column containing different solid materials. The mixture of proteins is forced through the columns by a pump rather than gravity. Therefore, the columns can be much longer and more densely packed, which results in a better separation of proteins.
Mass spectrometry for protein identification
Proteins can be identified through mass spectrometry, where the two most important ones are matrix-assisted laser desorption-ionization (MALDI) and electrospray ionization (ESI). MS measures the mass to charge ratio (m/z) of ions and allows derivation of the molecular weight.
The MS method generates gas-phase ions from an ion source (proteins), and these are separated based on size and charge (m/z). These patterns are detected, and are unique for each protein. Thus, the protein can be identified.
MALDi/TOF: Matrix-Assisted Laser Desorption-Ionization/Time-of-Flight
Type of mass spectrometry in which gas-phase ions are generated from a solid sample by a pulsed laser. The proteins are crystalized in a solid matrix and exposed to a laser. The laser excites the matrix, which transfers the energy to the crystallized protein. The energy then releases ions from the proteins, and the charge and size of these ions are unique for each protein. The ions travel along a vacuum tube, passing through a electric grid, which helps separate them by size and charge. The time-of-flight (TOF) detector measures the time for an ion to fly from the ion source to the detector. The TOF is proportional to the square root of their mass to charge (m/z) ratio. The molecular weight of the proteins can be determined from this data.
Electrospray ionization (ESI)
Type of mass spectrometry in which gas-phase ions are generated from ions in solution. A liquid sample of the proteins is put in a capillary tube, which allows droplets of liquid to emerge into a electrostatic field. The solvent evaporates and the droplets break up. Repeated evaporation and splitting of droplets eventually releases separate ions with different sizes and charges that are accelerated and separated towards a mass analyzer by an electric field. The pattern of these ions is unique for each protein.
An advantage of ESI is that it can be directly coupled to liquid separation techniques such as capillary electrophoresis or HPLC. Also, a parent ion can be isolated and fragmented into daughter ions, allowing more detailed analysis of the molecules, this is know as tandem mass spectrometry (MS/MS).
Tandem mass spectrometry (MS/MS)
Two successive rounds of mass spectrometry in which a parent ion is first isolated and then fragmented into daughter ions for more detailed analysis. It allows two parent ions with the same mass (e.g., two peptides with the same amino acid composition but different sequence) to be distinguished.
The MS/MS method generates gas-phase ions from an ion source (protein), and these are isolated according to their m/z ratio. These go through a reaction cell, and fragmented ions are separated based on their size and charge. These patterns are detected, and are unique for each protein. Thus, the protein can be identified.
Protein Interaction: Hybridization Systems
Proteins are screened by two-hybrid analysis to see which proteins interact/bind to each other in the cell. The two-hybrid system is a method of screening for protein-protein interactions, that uses fusions of the proteins being investigated to the two separate domains of a transcriptional activator protein. The test proteins (bait and prey) are fused separately to the two halves of a transcription factor. If the bait and prey bind each other they will reassemble the transcription factor and activate the transcription of the genes it controls. Two-hybrid analysis can also be used in mass screening by mating.
RNA three-hybrid systems identifies proteins that interact through an intermediary RNA molecule.
Protein Interaction: Co-Immunoprecipitation
A method of identifying protein-protein interaction by using antibodies to one of the proteins. An antibody which binds to the protein of interest is added to a mixture of multiple proteins. Beads coated with protein A, which binds the specific antibody, is then added. The bead complexes (bead with protein A, antibody, protein of interest and interacting proteins) are spun out, precipitation. The precipitation is ran on a SDS-PAGE to identify the proteins interacting with the protein of interest.
If two proteins are associated in a cell and one is precipitated by an antibody, the other should accompany it.
Protein arrays
Protein arrays are built using tagged proteins and are screened by a variety of approaches. His6 tagged proteins are bound to a glass slide with attached Nickel ions through their His6 tags, assembling a protein microarray.
Protein microarray is a microarray of immobilized proteins used for proteome analysis and normally screened by fluorescent labeling. To identify proteins that bind to ex. phosholipids, biotin-bound phospholipids are added to the protein microarray. Proteins binding phospholipids will now have biotin attached to them. Avidin with Cy3 fluorescent label is then added, and binds to the biotin, and the phosholipid-binding proteins can be identified.
