Protein

Question 1

Describe the hierarchy of protein structure

Answer 1

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

Question 2

What is mRNA and its relationship with protein levels in cells?

Answer 2

• 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

Question 3

What techniques can be used to quantify SINGLE protein in a cell?

Answer 3

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

Question 4

Define and give examples of post-translational modification:

Answer 4

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

Question 5

What are BCA assays and how do they work?

Answer 5

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

Question 6

What techniques can be used to quantify MULTIPLE protein in a cell?

Answer 6

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

Question 7

Describe the process of mass spectrometry

Answer 7

• 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

Question 8

What is proteomics?

Answer 8

The large scale study of proteins and their structure and function