Proteomics Flashcards
define proteomics
Study of structure, expression and interaction between proteins
what is the proteome
set of expressed proteins in a given type of cells /organism at a given time under defined conditions.
4 databases with protein annotations
UniProtKB - checked computationally, low quality
SwissProt- high quality, entries manually checked.
Prosite
Ensembl
way of understanding a protein
string of domains with own structure, function and evo history
what is ExPASy
proteomics server
has many databases
How are unknown proteins annotated computationally?
- analyse primary seq, compare to known domains/proteins.
- To predict 2/3 structure, probable sites of modification, probable functional properties
- scan 1 seq for known consensus signatures
what is a consensus signature?
certain structures which form clusters of related, characterised proteins.
how can we see the physical structure of proteins
Crystallising it
deduce AA seq and which structures they give rise to.
What is HMM
Hidden Markov Model
gives a probability score of how likely a sequence is to give a specific protein.
Forms consesus seq.
draw a simple HMM
.
What data would predict an a helical structure
.
structure prediction of TM domain
/
structure prediction of phosphorylation site
.
how does 2D PAGE work?
2D Polyacrylamide Gel Electrophoresis
Depends on charge and mass of proteins.
isoelectric focussing (gradient of pH across gel, proteins move and stop when at their isoelectric point. relies on AA amphipathic structure)
then separation by mass.
image gel and comp analysis can determine differences in dots on gel between samples.
What are the advantages of a 2D-PAGE?
Reproducible separation of protein mixtures
Identification of specific proteins
Allows qualitative comparison of the proteome
Comparably simple
What are the limitations of a 2D-PAGE?
Needs highly standardized procedure for accurate protein identification.
Databases needed for comparison with known proteins for identification
=> cannot identify previously unknown proteins
why is affinity chromatography for purifying one group /one protein like a fishing exp?
uses bait proteins coupled to a bead.
Can extract an unknown protein using a sunstrate for a desired active site eg.
describe Co-immunoprecipitation affinity chromatography
Mis protein mixture with antibody specific to POI. then run through column and immobilized protein binds to antibody. Also catches proteins in cell lysate which are interacting with antibody.
Elute.
describe Biotin- affinity chromatography
Biotin - small molecule, can be derivatized and attached to a molecule.
Avidin or Streptavidin (immobile) can bind specifically to biotin.
describe GST-fusion affinity chromatography
recombinant protein made with GST.
GST binds tightly to reduced glutathione which is on immoble phase in column.
Advantages of Affinity Chromatography
Extraction of groups of proteins
Extraction of a specific protein
Allows extraction of previously unknown proteins with putative functions
What are the limitations of Affinity Chromatography?
Can extract protein but not give further information
Relies on subsequent analysis of the proteins
Basic steps in mass spec for characterisation ond ID of protein groups
- Digest proteins using protease (trypsin)
- separation of peptides by charge and mass, correlate back to protein
- detection of specific peptide mass
3 parts of a mass spec
- ionisation source - converts molecules into gas-phas ions
- Mass analyser - separates ions by m/z ratio.
- ion detector - current over ime = signal amount at each m/z value.
Camputer analysis in mass spec
Comp can assign an ID to all peptides in a mix using the m/z value.
contrasts observed and theoretical profiles.
limitation of mass spec
doesnt work on very complex protein mixes.
relies on previos sequencing and prediction of peptides in a proteome.
advantages of mass spec
Can identify known proteins in a mixture.
very powerful when coupled with separation techniques eg LC/MS/MS, 2D PAGE
tandem mass spectrometry
MS/MS
Two mass specs separated by a collision cell
The first mass spectrometer is set to pass just one m/z value
This ion enters the collision cell and collides with argon or nitrogen
KE of ions converted to vibrational energy and the ions fragment
The m/z values of fragment ions are then determined in the second mass spectrometer
How can specific proteins be visualised in tissues
use highly specific monoclonal antibodies, in immunohistochemistry, western blotting and assays like ELISA
Can see is the POI is present in a certian tissue at a certain time, can analyse co localization of proteins and quickly detect presence of certain proteins
ELISA
Enzyme-Linked ImmunoSorbant Assay
uses principle of antibody-antibody interaction
HIV screen
- partially purified inactive HIV antigens on ELISA plate.
- Add serum containing antibodies. If patient is HIV+, serum contains antibodies to HIV and binds to antigens on plate.
- anti-human immunoglobin coupled to enzyme, binds to antibodies on plate.
- chromogen added, changes colour due to enzyme attached to 2 antibody. plate read at 450nm
describe an in vivo screen for protein-protein interactions
Yeast two-hybrid screen
- interaction of hybrid proteins are screened in yeast, bacteria or cell lines using reporter gene expression
- uses GAL4 TF of yeast. split into 2 parts. each part attached to different proteins. If proteins interact, the two parts are brought together and TF is active, transcribes a reporter gene.
Advantages of the Y2H system
Allows a large amount of candidates to be tested
Using a matrix approach entire genomes can be screened (mating libraries)
drawbacks of the Y2H system
High false negative/positive rate
Labor intensive and time consuming using proper replication
Limited to protein-protein interactions
