Studying Proteins.docx

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University of Toronto Scarborough
Biological Sciences
Rene Harrison

Studying Proteins 1. What are osteoclasts/osteoblasts and what do they do?  Osteoclasts are a large multinucleate bone cell that absorbs bone tissue during growth and healing. Thus it degrades bones.  Osteoblasts are cells that secrete the matrix for bone formation. Thus they are responsible for bone formation as well as making collagens 2. How can a person study proteins?  Proteins are mainly studied by: a. Determining primary structure b. Investigating secondary/tertiary structure c. Investigating protein localization d. Investigating protein function  Many of these techniques involve the use of protein purification, which is chemical methods, usually based on a combination of chemical properties and physical properties, by which a desired protein is isolated from the other proteins with which it normally occurs. 3. How do biologist determine the primary structure of a protein?  When determining the primary structure of a protein, biologists either: a. Extrapolate from genomic sequence b. Manually sequence the primary structure  Both these methods requires protein purification 4. How do biologist determine the primary structure of a protein using the genomic sequence?  During the process of genomic sequencing, biologists extrapolate amino acid sequence from gene/nucleotide sequence using computer database of genomes  The 1980‟s and 1990‟s were considered the “age of genomics” since this was around the time of The Human Genome Project vs. Celera 5. Who was Craig Ventor and why is he significant during the 1990‟s?  Craig Ventor was the founder of the Celera Genomics in 1999; he was able to create a invention to sequence the human genome faster then what The Human Genome Project was doing but waited on the same day to release the sequence  Both his and The Human Genome Project were able to find approximately 300,000 proteins and these DNA sequences can now be found in the Genbank 6. What are the consequences that arise with extrapolating from Genomic Sequence?  Extrapolating from Genomic Sequence has 2 limitations/issues: I. Introns/exons  Exons – code for proteins  Introns – “intervening sequences” that don’t code for anything  These introns splices out of mRNA and will not correctly predict protein sequence II. Posttranslational modifications often cannot be predicted from genome  Proteolysis  Glycosylation 7. How do biologist determine the primary structure of a protein using manual sequence?  Manual sequencing arose during “the age of proteomics”  During the manual sequencing of the primary structure of a protein mass spectrometry is used in which sequencing the primary sequence of proteins involves: a. Purifying the proteins either by: i. Proteins separated from a complex protein mixture on gel ii. Or proteins purified using chromatography 8. How do you purify proteins during manual sequencing using a protein gel electrophoresis?  When purifying a protein during manual sequencing of a primary structure using protein gel electrophoresis biologists: a. Cell lysates are made from cells of interest b. Proteins are denatured by heat and put in tracking dye containing SDS  SDS is negatively charged and coats proteins and makes all the proteins negatively charged c. Proteins are separated on a gel based on size  Gel used is called polyacrylamide gel which is a porous gel with many holes in it  The smaller proteins (low kDa) will move through quickly and larger proteins (high kDA) will move through very slowly d. Proteins move through by electrophoresis which is an electrical current which is applied through the gel so there is a positively charged end (anode) and a negatively charged end (cathode)  Proteins are all negatively charged thus they will all move towards the anode e. This is the 1-D gel electrophoresis  For 2-D gel electrophoresis, the proteins are first separated using isoelectric focusing based on the charge of the proteins  1000‟s of proteins can be resolved using the 2-D gel electrophoresis Figure 18.28 – Polyacrylamide gel electrophoresis. The protein prevents the sample from mixing with the buffer and then loadedsity into the wells with a fine pipette as shown in step 1. In step 2, a direct current is applied across the gel, which causes the proteins to move into the polyacrylamide along parallel lanes. When carried out in the detergent SDS, which is usually the case, the proteins move as mass. Once electrophoresis is completed, the gel is removed fromr the glass frame and stained in a tray step 3. Figure 18.29 – Two-dimensional gel HeLa cell non-histone chromosomal proteinsof labeled with 35Smethionine. Over a thousand different proteins can be resolved by this technique. 9. How do you purify proteins during manual sequencing using chromatography?  Biologist purify protein during manual sequencing using chromatography by first isolating proteins from their cell lysates and then the lysates are poured into column packed with beads  There are 3 major kinds of chromatography: a. Gel-filtration chromatography – which separates proteins based on size thus beads have different pore sizes and separates based on similar concept gel electrophoresis thus big molecules come out first Figure 18.25 – Gel filtration chromatography. The separation of three globular proteins having different molecular mass, as described in text. Among proteins of similar basic shape, larger molecules are eluted before smaller molecules. b. Ion-exchange chromatography – which separates proteins based on charge of the proteins since the gel resin has a charge separation of two proteins by DEAE-cellulose. In this case, a positively charged ion exchange is used to bind the negatively charged proteins. c. Affinity chromatography – which is the most specific method of the 3 methods discussed above and it exploits known protein affinities  Example: affinity between a receptor and ligand  Put the receptors on agarose beads and the lingands will bind to them and you can do vice-versa  OR you can put antibodies on agarose beads and target proteins antigens will bind doing this biologists can achieve pure protein purification Figure 18.26 – Affinity chromatography. (a) Schematic representation of the coated agarose beads to which only a specific protein can combine. (b) Steps in the chromatographic procedure. In and then in Fraction 2: receptors come outt first 10. How do you study protein sequencing using mass spectrometry?  In order to study protein sequencing using mass spectrometry you must first sequence the primary sequence a. Protein of interest must be cut out from 2-D gel and gel mixture must be removed ~ or you can use chromatography b. Proteins are then chopped into smaller peptide sequences using trypsin (protease)) c. Peptides are converted to an ion from and fired through a magnetic field this will separate in magnetic field at particular rate depending on their mass d. Then this will be detected by a spectrometer and it will measure the time in flight and can determine amino acid composition Figure 18.30 – Principles of operation of a mass spectrometer. Figure 2.49 – Identifying proteins isolated from a source (such as one of the spots on one of the gels of Figure 2.48) and subjected to digestion by the enzyme trypsin. The peptide fragments are then introduced into a mass spectrometer where they are ionized and separated according to their mass/charge (m/z) ratio. The separated peptides appear as a pattern of peaks whose precise m/z ratio is indicated. Figure 2.48 – The study of preoteomics mixtures of proteins. The two of complex electrophoretic gels shown here contain
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