Describe the hierarchy of protein structure
Are an essential part of all living organisms, can exist in one of 4 different structures, ranging from primary to tertiary
Primary - single chain of polypeptides (peptide bonds)
-> basic amino acid structure
DRAW AMINO ACID STRUCTURE
Secondary - more complex structures, alpha sheets and beta helixes, peptide bonds + hydrogen bonds between amino acids that are 3-5 positions apart
Tertiary - involve disulphide bonds between cystine residues of amino acids which are 10-15 positions apart
Quaternary - most structurally complex, hydrophobic interactions, hydrogen bonds and NON-covalent bonds
These are OLIGOMERIC proteins with 1+ polypeptide chains e.g. DNA, haemoglobin
What is mRNA and its relationship with protein levels in cells?
- messenger RNA
- essential in cellular replication as it is read in codons at the ribosome and tRNA brings the appropriate amino acid along to convert mRNA into a protein chain
- relationship can be positive/negative/neutral
- TRANSLATIONAL EFFICACY describes the quantity of protein produced from a mRNA transcript
- 60 codons code for ~20 amino acids
- some mRNA molecules are non coding, and some code for more than one amino acid
- proteins can also undergo post-translational modifications
What techniques can be used to quantify SINGLE protein in a cell?
ELIZA
- used to detect single protein from a liquid based sample
- uses 2 antibodies, the second of which undergoes a colour change
- commonly used in bowel cancer screening where it detects specific protein in stool of CRC patients
Gel electrophoresis (SDS-PAGE)
- protein treated with SDS (sodium dodecyl sulphate) to denature and unfold them into straight chains
- current passed through polyacrylamide gel and individual proteins travel further depending on their molecular weight (ratio of mass to charge)
- staining or another technique (western blotting) is then used to visualise the proteins separated on the PAGE gel
IHC
- similar to ELIZA, but this time the protein of interest is not in a liquid sample but is fixed in paraffin/solid
- allows quantity and location of protein to be found
- slides with microtomes undergo antibody soaking and then staining
Define and give examples of post-translational modification:
Changes which occur to proteins after they have been made at the ribosome, includes:
- phosphorylation
- ubiquitination
- acetylation
- methylation
PTM make it difficult for proteins to be detected by mass spectrometry if you do not know their mass as you cannot identify them
What are BCA assays and how do they work?
Bichinchoninic acid assay
Biochemical assay to determine CONCENTRATION of one protein out of a liquid solution
BCA is added and causes chelation between peptide bonds in the protein and the copper ions in the BCA
Chelated copper ions undergo green -> purple colour change, and wavelength of light emitted from the sample quantifies how much protein is present
E.g. to detect protein in urine
What techniques can be used to quantify MULTIPLE protein in a cell?
Microarrays
- antibody array packs can be pre-bought
- contain wells with different antibodies that will bind to different classes of proteins e.g. ‘proliferation’ proteins or ‘metabolism’ proteins
- liquid solution containing protein of interest is poured over the tray of antibodies
PAPER: Nordstrom et al 2014 -> used microarrays to detect protein biomarkers for stratifying prostate cancer into high/low risk groups as PSA is not a reliable marker
Mass spectrometry
- analytic tool used to measure the molecular mass of proteins
- proteins ionised and accelerated through electric field where individual mass and charge are identified
PAPER: Wilcken et al 2007 -> to detect higher ratio of the amino acid phenylalanine:tyrosine and to diagnose PKU in babies from blood spot
2D-DiGE (2D difference gel electrophoresis)
- seperates out a mixed sample of proteins to allow individual protein identification
- protein sample exposed to isoelectric current first which separates them based on pH
- then undergo normal electrophoresis to separate out the protein of same pH by their size
Describe the process of mass spectrometry
- it is an analytical tool for measuring the molecular mass of a sample
- ionised biomolecules accelerated through an electric field
- ionised molecules are seperated according to their mass/charge ratio
- each molecule releases a distinct signal as it is accelerated through the tube, and this is detected using a computer
Often the desired proteins cannot be detected using Mass spec, as their actual mass after they have undergone post-translational modifications are not known
What is proteomics?
The large scale study of proteins and their structure and function
