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Protein sequencing
Uni of Notts, Structure Function & Analysis of Proteins, year 2, topic 5
| Term | Definition |
|---|---|
| How number of subunits in a homomultimer is determined | Divide native molecular weight by denatured molecular weight to estimate number of identical subunits. Can't be done in heteromultimers |
| How molecular weight is estimated during gel filtration | Plot log(MW) vs volume of elution solution needed for standards to flow through, then use curve to estimate unknown protein size by plotting elution volume needed for each unknown sample |
| How SDS-PAGE standard curves estimate protein size | Log molecular weight (Y axis) vs migration (X axis) distance to estimate unknown protein size. Calibration curve is formed using the standards |
| Edman degradation | Classical protein sequencing technique which sequentially removes & identifies N-terminal amino acids to determine protein primary structure |
| Issues with Edman degradation | Limited to short peptides (20-30AA) & slower than mass spectrometry-based sequencing methods |
| Peptide mass fingerprinting (PMF) | Identifies proteins by excising protein gel spots & hydrolysing primary structures using trypsin to form tryptic peptide fragments then matching these to database patterns |
| Why PMF is rarely used | Requires known database matches & lacks detailed sequence information |
| How PMF tryptic fragments are generated | Trypsin cleaves after every basic amino acid (arg or lys) toward the C-terminal side. They're protonated then run through mass spectrometry to determine their mass:charge ratio |
| Approximate size of tryptic peptides | ~1-2 kD since lys & arg make up roughly 10% of amino acids due to each of 20 amino acids having roughly the same probability of being used |
| Tandem MS | Modified PMF. Fingerprinted tryptic peptides moved to collision chamber & collided with nobel gasses to progressively shrink them to smaller fragments & difference in mass is measured as peaks to determine mass of amino acids lost |
| How to determine differences between amino acids with identical weight (glutamine & lysine, leucine & isoleucine) in Tandem/MS | Lysine is negatively charged so will only be found at N-terminus. Leucine & isoleucine can't be distinguished using tandem-MS |
| Y-series | Nobel gasses can cleave any bond (a, b, c for N-terminus & x, y, z for C) but only b & y cleavages leave daughter ions with exposed C-termini to contain protons which can be measured by MS |
Created by:
Denny12
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