lecture 4 biochemistry (week 1) Flashcards
(17 cards)
what is proteomics?
describes the research focussed on the analysis of large sets of proteins
analogous to the term genomics
relies on affinity tagging and mass spectrometry
can determine the direct binding interactions between many different proteins in a cell
resulting in databases of protein interaction maps
what is Skp1?
S-phase kinase associated protein
part of the SCF ubiquitin ligase
functions as a protein complex to catalyse ubiquitination
has multiple functions out with ubiquitination - functions at the origin of replication, in cell cycle progression, in methionine synthesis, and in the kinetochore
what is the protein interaction map?
when hundreds or thousands of proteins are displayed on the same map the network diagram becomes bewilderingly
complicated
tends now to be smaller sub-sections of these maps that are considered, focussed around a few proteins of interest
what is the overview of the proteomics workflow?
sample preparation - whole extracts, protein complexes by precipitation (immuno-, affinity-, bait proteins etc.)
sample separation - labelling/staining
spot picking, polypeptide band selection
trypsin digestion - generation of peptide fragments
mass spectrometry
data analysis - protein identification
how can samples be prepared?
whole cell extracts - standard lysis with laemmli buffer
protein complexes by precipitation - immuno-precipitation (using antibody to protein of interest)
affinity based capture (GST-glutathione, biotin-streptavidin etc)
use of overexpressed bait proteins etc (eg. GFP tagged, GST tagged, HA tagged baits)
generates a subset of proteins: those interacting with target protein of interest
how else can samples be prepared?
labelling by staining - coomassie brilliant blue R-250 (dye-based)
colloidal coomassie blue G-250
silver (silver ions binding to protein; hazardous, not compatible with mass spec.)
sensitivity of silver is down to about 0.3ng vs >7ng for coomassie however, colloidal coomassie -250 offers a compromise as good sensitivity to approxiamately 1ng/band
colloidal coomassie G-250 used routinely in proteomics
some approaches now utilise labelling pre-
separation e.g. DIGE methodology (GEHealthcare)
uses labelling of different samples with different fluorescent dyes
CyDye™ DIGE fluors for protein labelling – separate proteins by 2D and image
what is isoelectric focussing?
a method of separating proteins according to their isoelectric points in a pH gradient
IP is defined as the pH that the proteins carries no net charge, or which proteins become immobile in an electric field
describe 2D electrophoresis after IF?
the pH gradient of the electric field the electrophoretic separation is used in polyacrylamide gel (SDS-PAGE)
proteins can be visualized after staining
what is polypeptide band selection?
polypeptide band excision - post 1D electrophoresis - cut out band of interest with scalpel
post 2D electrophoresis - spot picking - eg. GEHealthcare Ettan Spot Picker: automatically picks selected protein spots from stained or destained gels
what is trypsin digestion?
generation of peptide fragments; from intact proteins to peptides by in-gel digestion
varied size based on presence of trypsin sensitive cleavage site amino acid specific
what is mass spectrometry?
enables the precise measurement of molecular weight of a broad spectrum of substances
as the studied substance has to be intact in gas phase, the use of mass spectrometry for protein analysis was only enabled by development of soft ionisation
identification of peptide fragments
how can protein identification be generally performed?
Protein is digested by trypsin or by other proteolytic enzyme to smaller peptides and their precise molecular weights are measured using MS. The spectrum of those molecular weights is then compared with theoretical spectra that are calculated from protein sequences from available databases
Tandem MS enables to choose the peptide which is then fragmented by the collision with inert gas. The fragmentation pattern gives either full of partial information about protein sequence that is subjected to the search in databases
As a part of protein identification, MS is a priceless tool for protein post-translational modification analysis (e.g. phospho-proteomics), because it enables to localise given modifications within the protein and also helps to find out the nature of such modification
how does data analysis occur?
drive toward identification of proteins
sequences of amino acid fragments
input into protein sequence database(s)
examples of data bases…MASCOT…..ExPASy etc.
what occurs in post analysis?
validation of results
changes in expression - spot size of tissues
changes in protein complexes as protein-protein interactions: control vs stimulated -
small scale immuno-precipitation/’pull-down’ experiments with a focus on one or two proteins
bi-directional analysis: each partner as ‘bait’
what are the applications in health and disease: qualitative and quantitative?
HEALTHY VS. DISEASED
CONTROL VS. STIMULATED
OVER-EXPRESSED (TRANSFECTED) VS. NORMAL
WILD-TYPE VS. KNOCK-OUT
what are the goals of proteomics in medicine?
one of the main goals of proteomics in medicine is the identification of novel markers that can be used for prediction, prevention, diagnosis, prognosis and therapy optimisation in human disease
an aim to use proteomics in real everyday clinical practice it is necessary that the biological material where such marker would be analysed is easily accessible (blood, urine, saliva, cerebrospinal fluid).
used mainly in experimental settings with models of disease; one of the emerging applications is in the field of therapeutic drug discovery and pharmacoproteomics.
