Lecture 9a Electrophoresis/ Mass Spectrometry

Question 1

Using light absorbance to measure/ follow reaction rates

Answer 1

Many enzymes do not use substances that change absorbance in a usable wavelength range so synthetic substrates designed to produce change in absorbance may be used

E.g. proteolytic enzyme chymotrypsin can be assayed using a synthetic substrate containing a compound that absorbs in visible region on hydrolysis

Question 2

Fluorescent proteins

Answer 2

Jellyfish Aequora victoria have green fluorescent protein. This is due to the aequorin enzyme that catalyses a reaction causing blue light and green fluorescent protein that generates green fluorescence on blue light excitation.

Green fluorescent protein widely used for probe or reporter in mol and cell bio

Can be expressed in transgenic organisms to produce fluorescent phenotypes that can be used to characterise cell-cell gene expression

Using invitro mutagenisis and recombination protein technology a range of modified fluorescent proteins with range of colours have been produced widening potential use

Question 3

Light absorbance/fluorescence summary

Answer 3

Absorbance and fluorescence of UV and visible light results from excitation of electrons in molecules to higher excitatory levels

Simple line spectra is broadened/complicated by molecular motion (vibration/rotation/translation) and interactions with solvent molecules

Leading to broad peaks in absorbance and emission where Maxima is fixed by electronic transition.

Absorbance in solution proportional to conc. Of absorbing compound - allows us to determine conc. Of compounds

Fluorescence results from emission of light instead of dissipation of absorbed energy by molecular motion - a property of specific compounds

Fluorescence is useful for imaging and detection - availability of a range of highly sensitive detectors for emitted light available today

Fluorescence in solution is proportional to concentration of fluorescent compound

Question 4

Electrophoresis

Answer 4

Separates proteins by molecular weight, charge and isoelectric point

Can use agar heat activated or acrylamide chemical activated to make a gel from powder.

Used for protein and nucleic acid analysis

SDS sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) process:

Polyacrylamide gel slab prepared

Protein mixed w/SDS that acts as a detergent (e.g. SLS)

Proteins binding to hydrophobic residues form a complex where SDS bound per amino acid residue is nearly equal

SDS denatures

Reduced to remove disulfide bonds

Behave like linear molecules on electrophoresis

• SDS swamps protein charge so migration is solely due to size of polypeptides

Question 5

SDS PAGE - why popular choice?

Answer 5

One of the most widely used protein analyses, simple, cheap, sensitive to a microgram of protein (10-⁶g) or even ng (10-⁹g) with suitable stains, also quick

Determines polypep weight not protein molecular weigh

Question 6

Western blotting

Answer 6

Proteins on a gel are transferred to a membrane by passing an electric current at right angle through gel sheet

Uses antibodies to detect a protein - an antibody binds to target protein and secondary protein binds to this enzyme. When linked to the secondary antibody a visible colour change (blot) is produced

Question 7

Isoelectrofocussing

Answer 7

Gel 1 runs samples in pH gradient low to high

Proteins migrate to isoelectric point (where they are neutral)

Gel 1 is placed on SDS polyacrylamide slab and run to separate polypep. weights

Allowed 1000 proteins to be identified in E. coli sample

Protein spots can be extracted for analysis

Question 8

Mass spectroscopy : Malditof

Answer 8

Superceded isoelectrofocussing
Identifies proteins based on sequences specific clearance into fragments

Matrix assisted laser desorption ionisation time of flight spectrometer aka Malditof mass spectrometer

Malditof mass spectrometer used to measure polypeptide weight

1) put proteins on surface
2)add inorganic compound to surface to form crystals
3) irradiate crystals with a pulse of powerful laser
4) proteins will be blown off (desorbed/ionised)
5) proteins sucked into mass spectrometer vacuum
6) protein identified

Question 9

Electrospray ionisation (ESI)

Answer 9

SDS PAGE protein band cut from sheet

Unknown protein digested by trypsin

Protein fragments aka tryptic peptidase

Put into TOF MS for analysis

Identified via data base search of trypsin

Protein can be identified by polypeptide fragment fingerprints

Question 10

Protein sequencing

Answer 10

Can be determined directly or predicted from encoding gene

Sequencing comparisons unform evolutionary links

Direct determination of protein sequence requires large quantities of purified proteins

Based on biochem techniques
Earliest technique used N terminal sequencing via Edman degradation (used to compare proteins from diff organisms)

Edman degradation- amino acid residues chemically removed from N terminus of protein one at a time and identified by chromatography.
Possible to identify 50 aminos in 24 hrs but then it would have to be stopped

Question 11

Edman degradation

Answer 11

Edman degradation- amino acid residues chemically removed from N terminus of protein one at a time and identified by chromatography.
Possible to identify 50 aminos in 24 hrs but then it would have to be stopped so overlapping fragments had to be sequenced. So this was very time consuming.

Question 12

Mass spectrometry

Answer 12

Also used protein sequencing (proteomics) is most common method for directly sequencing a protein

Question 13

DNA sequencing

Answer 13

Currently most used

Fast and easy, uses molecular biotechnology. Readily automated so current machines can determine an entire bacterial genome (~5million base pairs) in 2hrs.

There are more predicted protein sequences based on DNA than on direct protein sequences

Question 14

Edman degradation process

Answer 14

1) break down protein into fragments by protease
2)purify fragments - determine sequence of fragments by removing N terminal residues one at a time.Each amino elutes at defined time from chromatography column
3) assemble fragments into complete sequence

Question 15

Mass spectrometry (MALDITOF)

Answer 15

1) break protein into fragments with protease

2) ESI MS to separate/identify fragments by mass: liquid>electrospray needle> electrospray ionisation: converts spray to droplets containing soluble ions - Liquid around ions dries as they are sucked into the machine

3)Sub-fragmentation of fragments retained in ‘ion trap’ identify amino residues

4) compare sequence of fragments
To protein DNA sequence database to identify proteins

Question 16

Protein sequencing provides

Answer 16

Amino sequence related to an encoding gene for ID comparison

It shows post transcriptional modifications and disulphides that cannot be seen in DNA sequencing

Question 17

Orthologues and paralogues

Answer 17

Sequence comparisons:

Orthologues: info regarding evolution distance between organisms

Paralogues: evolution link derived between diff proteins

E.g. human and bovine ribonuclease are orthologues - same function/diff origin

Human ribonuclease and angiogenic are paralogues - same organism diff function

Proteins w/similar structure likely to have similar functions

Question 18

Phylogenetic research

Answer 18

T-coffee
Muscle - sequence alignment
Clustal omega

Multiple sequence aligners form phylogenetic trees

Divergent radiation from common ancestor

Convergent evolving to same function from diff ancestors

Question 19

How are protein structures determined?

Answer 19

Research began in 1950’s with X ray crystallography
- X ray crystallography has been used to identify 90% of extant structures

Nuclear magnetic resonance spectroscopy used for small, flexible proteins

Electron microscopy for large protein - provides overall shape but res approaching X ray cryst uses cryo- EM which challenges X ray cryst for utility

~1mg protein required for identification of structure and this size is reducing w/crypto EM

Protein structure predicting software is not yet reliable but Alpha ‘fold’ AI is usually correct

Question 20

X ray crystallography

Answer 20

X ray source projects a beam onto a protein crystal, the diffracted beam is collected on detector film

Diffracted rays detected as a series of dots, from position and intensity of dots the spacing of atoms in the crystal can be deduced and interpreted like a map
Of electron density into which aminos fit

It is a slow and complex process requiring the protein to be crystallised - many proteins cannot be crystallised and dynamic structures cannot be observed

Question 21

NMR nuclear magnetic resonance spectroscopy

Answer 21

Done in solution to look at dynamic structures. It is non- invasive and requires a large quantity of protein. Useful to see flexibility, limited to small proteins

Question 22

Electron microscopy

Answer 22

Can show low resolution images of large molecules such as protein complexes

Resolution of cryo EM now as good as X ray crystallography, good for large complexes with no need for crystallisation and computerized so much faster - takes only a few hours.

Cryo: Electrons projected onto flash frozen sample on a grid, magnetic lense beneath forms an image

Neg. Stain method: same but sample coated in heavy metal