Electrophoresis and mass spectrometry Flashcards
2 important factor in the mobility of proteins
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
SDS PAGE for protein separation
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
discontinuous gels
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
dyes
Coomaisse - stick v lightly to proteins through hydrophobic interactions
2D gel electrophoresis
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
at ph7.4
aspartate and glutamate will e negative
lysine and arginine and histidine will be positive
isoelectric poin
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
iselectric focussing
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
- load evenly across IPG strip
- focus high voltage
proteins focus an remain at a position in the matrix equivalent to their isoelectric value
protein identification
- fragmentation
- mass spectrometry
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
Edman degradation protein sequencing
coupling reagent - edman reagent react with the N terminus which cyclises and selective cleavages polypeptide chains and coverts into product which and be analysed
- Coupling, add reagent to label the N-terminal AA
- Cleave the first peptide bond (only) to release labelled AA
- Identify released AA
- Recover (n-1) peptide for 2nd round
- Repeat on recovered peptide to find 3rd residue
easy to get errors in identification
months for full sequence identification
swissprot database of protein sequences
human + mice
genbank
dna sequences database
Accurately determine the molecular weight of a sample
- ion source
MALDI/ ES
Converts molecules to gas phase ions each with unique mass/charge (m/z) ratios - mass analyser
TOF/ quadrupole
Separation of individual ions by mass/charge (m/z) properties - 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.
Protein identification by Tryptic fingerprinting
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
MALDI Matrix-assisted laser desorption ionisation mass spectrometry
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
ionisation
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
Quadrupole is an ion gate mass filter.
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.
Protein identification mass spec sequencing
ES Q-TOF mass spectrometer (MS/MS)
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.
RNA splicing-removal of introns
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
He La-s3
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
SAGA purification
yeast cell extract
9 Columns
- Anion exchange
- Cation exchange
- Histone affinity
- DNA affinity
- Gel filtration
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
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
methodology improved eliminate false positives by:
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
yeast interactome
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
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
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
