Electrophoresis and mass spectrometry

Question 1

2 important factor in the mobility of proteins

Answer 1

seperation by sie alone:

Buffers ussed ofr gel & reservoirs i.e. Tris-edta

Double stranded DNA
agarose of acrylamide

single stranded agaorise or acrylamide with formamide or urea

control of porosity is the conc of acrylamide chains higher ratio bigger the size - protein as opposed to DNA

sizing dna fragments: then
smaller the amount of DNP base pairs the the higher acrylamide percentage

log of molecular weight = proportional to the distance travelled

Question 2

SDS PAGE for protein separation

Answer 2

determine molecular weight of molecular proteins

anionic detergent - a sulphate group on on a hydrocarbon chain

interacts w water

hydrophobic tails into hydrophobic regions of the protein: disrupts hydrophobic interactions
within protein core
between proteins in a vomplex
between proteins and membrane proteins

1 SDS for every 2 amino acids

natural charge samped all proteins become positive
charge proportional to length
protein denatures an fritictional drag is proportional to length
in free sol all proteins have same mobility
by choosing right gel matrix according to size of porous molecular sieving effects separate proteins on the basis of molecular size alone

Question 3

discontinuous gels

Answer 3

electrode buffer
ph8.3
tris glycine mixture containing SDS

stakcing gel ph g.8- low conc of acrylamide contains SDS

resolving gel ph8.8
where the seperation of components takes place

so start off at ph8.3 everything moving from negative to positive as soon as hits glycine becomes ph 6.8 its net charge will be zero so glycine slows down and become non conduction as it is a xwitter ion equally pulled in both directions - creates a disproportionate distribution of the electrical potential over the gel in that zone
voltage shoots up over
= ions all stack up behind each other by the time it reaches separation gel they are all lined up
at ph8.8 glycine becomes negatively charged again

= stacking generates resolution

low percentage gel produces large pores

gel buffer cloride ions from tris hcl form an ion infront ahead of sample zone

electrode buffer ions glycine from tis glycine form a zone behind the sample

proteins all with the same electrophoretic mobility are sandwiched between the fast moving gel buffer cloride ions and sow moving reservoir buffer glycine ion fronts

low conductivity in stacking region

lower the protein size in kDa the lower the acrylamide conc

low pH proteins have a strong +ve charge = reat w die

Question 4

dyes

Answer 4

Coomaisse - stick v lightly to proteins through hydrophobic interactions

Question 5

2D gel electrophoresis

Answer 5

fractionate protein or an immobilised PH gradients
gel is desalted

proteins will move off
when they reach a ph where their net charge is 0they will stop moving
pu tonto another gel and run SDS page in other dirrection

separating protein with respect to 2 parameters
isoelectric focusing and SPS
charge and molecular weight

Question 6

at ph7.4

Answer 6

aspartate and glutamate will e negative

lysine and arginine and histidine will be positive

Question 7

isoelectric poin

Answer 7

the pH at which there is a prcise balance between the positive and negative charges

protein have 0 net charges

charge on amino acids changes depending on pH due to the titration of the charged side chain groups

in electric field negatively charged ravel towards anode untilthey reach isoelectric point

= can fractionate protein mixture with respect of the isoelectric point of all the proteins as proteins have different pI values
i.e pepsin is lower than 1 and lysozyme = 11

Question 8

iselectric focussing

Answer 8

a protein in a buffer of pH equal to its pI has a net xero charge and will not migrate in an electric field

in IEF proteins move in both directions to focus at their pI

use immobilised ph gradient strips

1. load evenly across IPG strip
2. focus high voltage
   proteins focus an remain at a position in the matrix equivalent to their isoelectric value

Question 9

protein identification

1. fragmentation
2. mass spectrometry

Answer 9

1. SEQUENCED GENOME 
KNOWN PROTEIN
SEQUENCE NOT REQUIRED
MALDI ToF - protein identification
matrix assisted laser desorption ionization - time of flight
Peptide fingerprint
Database search

2. PARTIALLY SEQUENCES GENOME 
REQUIRE SEQUENCE:
Tandem mass spec (MS/MS)
Peptide seqencing
Database search

ESQ-ToP - protein sequence determination
electrospray quadrupole - time of flight

weighing molecules in gas phase with remarkable accuracy

Question 10

Edman degradation protein sequencing

Answer 10

coupling reagent - edman reagent react with the N terminus which cyclises and selective cleavages polypeptide chains and coverts into product which and be analysed

1. Coupling, add reagent to label the N-terminal AA​
2. Cleave the first peptide bond (only) to release labelled AA​
3. Identify released AA ​
4. Recover (n-1) peptide for 2nd round​
5. Repeat on recovered peptide to find 3rd residue

easy to get errors in identification

months for full sequence identification

Question 11

swissprot database of protein sequences

human + mice

Answer 11

genbank

dna sequences database

Question 12

Accurately determine the molecular weight of a sample​

Answer 12

1. ion source
   MALDI/ ES
   Converts molecules to gas phase ions each with unique mass/charge (m/z) ratios​
2. mass analyser
   TOF/ quadrupole
   Separation of individual ions by mass/charge (m/z) properties​
3. ion detector
   Ions strike the detector and yield a current. The magnitude of the current as function of time is used to determine the mass/charge (m/z) ratio.

Question 13

Protein identification by Tryptic fingerprinting

Answer 13

No sequence information required​

i.e. sequence present in the protein database ​
MALDI TOF

having degraded the unknown protein into fragments by trypsin cleavage (lys/arg) put into organic acid solid target fired the laser at it

ions accelerated in electric field
time of flight
mass analyser into ion detector
recording m/z

Question 14

MALDI Matrix-assisted laser desorption ionisation mass spectrometry​

Answer 14

Peptide fragments applied to solid matrix and irradiated by energy from laser pulse to either add or remove protons to generate positive ions​

Ions released are accelerated to a fixed amount of kinetic energy and travel down a flight tube. ​

Small ions have higher velocity and are recorded on the detector before larger ions.

compare with theoretical cut with trypsin from protein database

automated
MASCOT serves for peptide mass finger-printer

Question 15

ionisation

Answer 15

Peptide fragment solution is dispersed into highly charged droplets by passing through a needle under high voltage, 3000 V, electric field. Adds protons to generate ions The charge on carboxy and amino terminal fragments is retained so multiply charged ions can result

Question 16

Quadrupole is an ion gate mass filter.

Answer 16

Consists of 4 rods with to which an oscillating electric field applied allowing only ions with certain masses to pass through to the detector. Other masses are on unstable trajectories and do not reach the detector.
Electric field is adjusted to allow different masses to the detector (scanning) ​= spectrum​

can select individual ion for further analysis

ts mass/charge ratio is allowed​
particular ac/dc voltage settings on the Quad.​
​
The source is usually configured ​

to fly positive molecular ions but​

can be set for negative ions also.​

​

Question 17

Protein identification mass spec sequencing

ES Q-TOF mass spectrometer (MS/MS)​

Answer 17

Stage 1 - collection of full spectrum​
2D gel - spot is excised and bleached out of acrylamide gell
treated with trypsin to make peptide fragments
put into spectrometer
one of the ions is enabled to get through the
Quadrupole - enters collusion cell which contains
noble gas ions which collides causing ions to break into pieces each of which are analysed in the tube of the mass spectrometer at time of flight

= core info used for sequencing the peptide

Stage 2 - MS/MS​

Single ion chosen from spectrum for​

Sequence analysis.​

Ion passed into collision chamber containing an inert gas. Collision of ion with gas causes the fragmentation of the peptide ion. These fragments are detected by TOF mass analyser to produce spectrum.​

Question 18

RNA splicing-removal of introns

Answer 18

RNA and proteins are highly conserved in eukaryotes from yeast to human​

U1 snRNP binds to primary transcript​

Addition of U2,U4,U5 U6 snRNPs forms the spliceosome​

What is the nature of the snRNPs ?​

In particular U1 snRNP​

Question 19

He La-s3

Answer 19

sample fractionation :
cytosol / nuclear extracts

fragmented proteins by trypsin degradation

immuno affinity purification using antibody that was able to recognise acetylated lysine took out lysine peptides = subject to HPLC column
ran tandem mass spec analysis
pick out primary ions from initial scan
put into fragmentation cell
identify sequence of acetylated protein
acetylation has an effect on trypsin degradation no cleave at acetlated lysine site

Question 20

SAGA purification

Answer 20

yeast cell extract

9 Columns​

• Anion exchange​
• Cation exchange​
• Histone affinity​
• DNA affinity​
• Gel filtration​

Question 21

yeast 2 hybrid screen to look for 1 to 1 interactions

Positive​

Both approaches give large numbers of interacting proteins >500​

Indicative of large networks of interacting proteins​

Negative​

Only small number of overlapping interactions between the two techniques​

Yeast 2 hybrid approach can suffer from a high degree of false positives​

Answer 21

Gal-4:
dna binding domain + activation domain

when complex interacts with a histidine reporter gene yeast will grown on a medium that contains no histidine

gene not switched on yeast will not grow

2 domains can activate each other without contact

Clone ORFs into both bait and prey strains and mate ​together

Bait and prey can be from any organism e.g. yeast, drosophila, C. Elegans
bait-prey complex formed = brings activation domain into operation = swiches on histodine biosynthesis

Each colony represents a single protein-protein interaction

protein identity from positive colonies
- all respond to bait 
histodine biosynthesis taking place
primers 
sequence dna = pluck out what protein is corresponding to activation of  histidine biosynthesis 

Each positive colony on 2 hybrid screen gives a single interaction between two proteins.​

​

Interacting proteins are joined in an interaction map​

Question 22

methodology improved eliminate false positives by:​

Answer 22

comparison with experimental data (i.e. IP data)​

genetic interaction data​

gene expression data​

training sets, e.g. ​

nuclear-nuclear allowed​

cytoplasmic-cytoplasmic allowed​

nuclear-cytoplasmic less likely​

statistics- confidence level predicts interaction​

interaction maps automated process of drawing​

Question 23

yeast interactome

Answer 23

Clusters of highly connected nuclear protein complexes. The central densest region of a large interaction network containing over 15 000 protein interactions​

kinases are more dense than transcription factors

Question 24

Protein Kinase C (PKC) pathway- yeast cell integrity. The PKC MAPK cascade is activated in response to heat and low osmotic stress and nutrient limitation

Answer 24

Hcs77 is a putative mechanosensor that is proposed to sense membrane stretch.
The same proteins laid out automatically using protein–protein, protein–DNA and genetic interaction