Proteomics -

Strengths and Weaknesses

 What is Proteomics?
 Studying characteristics and activity of every protein
within the cell
 The proteome is the entire complement of proteins
which the cell expresses

You could also look at :

Genome – all genes encoded by DNA
Transcriptome – all mRNA transcribed from genome
 But protein levels and mRNA levels do not always correlate
 Why use proteomics?
Proteome best to look at because:
Single gene can encode several forms of a protein based
on splice variants, frame shifts, distribution within cell,
stability and post-translational modifications
Post-translational modifications include: phosphorylation,
glycosylations, acetylation, linkage to GPI anchors,
sumoylation, ubiquitination. There’s ~300 different
types reported
These modifications affect localisation, stability, binding
interactions, function
 Uses of proteomics
 Protein isolation
 Studying post-translational modifications
 Identifying Isoforms
 Protein-protein interactions
 Protein hunting for novel proteins
 Polymorphism studies
 General Pros and Cons
 In brain systems protein levels are mainly regulated by
proteolysis and protein stability rather than
transcriptional level
 Protein levels don’t always match with that of nucleic
acids
 Gives information about activation states and post-
translational modifications
 Steps in Proteomics

 Target identification
 Isolation
 Western Blotting
 Mass Spectrometry
 Crude Assembly
 Database searches
 Demonstration of interacting proteins
 Assembly of Complexes
 Dynamics
 Target Identification and isolation
 Not all proteins are expressed at levels detectable by
proteomics
 Problems with extracting membrane bound proteins
 High Mr proteins don’t get separated properly by
electrophoresis
 Low Mr proteins are difficult to identify after in gel
digestion as you get few peptides
 Post-translational modifications cannot be determined
during high throughput
 Western Blotting
 Can give definite answer •Protein must have known
as to whether a specific antibody to show up so
protein is present wont show novel proteins
 Very Sensitive, can detect •You must know what you
protein levels less than mass are looking for
•Labour Intensive
spectrometry can •Difficult to do on a large
scale
 Mass Spectrometry - Strengths

 Potential to do it automated, therefore large scale

 Shows post translational modifications eg. 80kDA is a
phosphate group
 Don’t need to know what you are looking for
 Mass Spectrometry - Weaknesses
 Can distinguish peptides with similar mass, but not the
residues Ile/Leu which have the same mass
 MALDI-TOF allows only 40-60% of spots from gel to be
identified
 Spots may contain more than one protein
 Novel proteins will not be listed in database
 Although tells you if peptide is phosphorylated, doesn’t
tell you where or when
 Database searching - Weaknesses
 Takes months
 No single database, need to use lots of them to look at
protein identification, sequence homology, protein-
protein interactions etc.
 Novel proteins not listed yet
Alternative Steps to improve results

 2D gels
 DIGE
 Liquid Chromatograpahy
 ELISA
 Phage displays
 Yeast two-hybrid studies
 NMR straight from a protein spot
 Powerblot
 2D gels
Separated first based Results vary between animals,
on isoelectric charge so many animals are needed
and then on and results are averaged
molecular weight Large or small peptides hard
to resolve

High Reproducibility Very acidic or basic peptides

hard to resolve
Higher resolution
separation – thousands Single spot can contain
of protein spots products of many genes and
the products of a gene may be
Fast contained in many spots
Easy to do Not quantative
 DIGE - difference gel
electrophoresis
 Look at several pools at once, but label each with a different
fluorescent marker and do subtraction of intensities

Can run two samples in one Doesn’t help with post-

gel translations as just
Easy to quantify shows difference in
levels
Doesn’t get in the way of
mass spectrometry
 Elisa – Enzyme Linked
immunosorbent assay
Use immobilised antibodies as an array to
capture specific peptides

 Easy to set up • Need to know what you’re

 Automated looking for
 Reproducible
 MudPIT
Alternative to 2D PAGE separation
2D separation based on liquid chromatography
Then coupled to tandem mass spectrometry

Automated
Proteins can be in solution phase
Multidimensional
Good for low expressed proteins
 Peptide Arrays
 Array of peptides expressed in bacteriophage
 Target protein coupled to membrane or chip then
exposed to the bacteriophages
 Interacting proteins can then be identified
Or
 Yeast two-hybrid system
 ‘Powerblot’ – Western Array
 Allows you to analyse for >800 proteins simultaneously
using a cocktail of antibodies each labelled with a
different fluorescence
 Slice the gel into lanes and then fluorescence is read by
an automated machine
 Can do this for diseased model and control and compare
protein expression
 Can include an internal control, such as actin, as the
levels of expression of cytoskeletal proteins remain fairly
constant
 ‘Powerblot’ – Western Array
 You can find things you  Cant distinguish cellular
weren’t even looking for distribution as lysate is
– ‘fishing’ everything mashed
 Shows changes in protein together
levels, not genes  Need large sample of
 Can distinguish between animals to give enough
activation states using lysate to work with (>9
specific antibodies mice)
 Wont show novel proteins
 FTICR Mass Spectroscopy

Fourier transform ion cyclotron resonance Mass Spec

Gives highest mass measurement accuracy so protein

identification can be based solely on mass of a peptide
fragment, so don’t need to carry out tandem mass spec

If you combine capillary Liquid chromatography with

FTICR you can get high throughput, very sensitive
method for identification and quantification
 ICAT – isotope coded affinity tag

Label cysteine residues with reagent containing a biotin

group which can be used to isolate the peptides which
are modified
Extracted peptides can then be analysed separately.
Allows low abundance proteins to be quantified as mixture
is less complex
2 separate labels – heavy and light ICAT so two samples
can be mixed and then separated again, or ratios can be
compared
 Phosphorylation studies
 Important Post-translational modification used to
modulate protein activity
 Label proteins with 32P and then do 2D-PAGE
 Compare relative intensities of various spots to quantify
phosphorylation levels
 Not useful for high throughput analysis
 Enhance studies by getting rid of non phosphorylated
protein in mixture using phospho-specific antibodies or
metal affinity columns
 Proteomics and the CNS

 Used to compare levels of proteins in studies looking at

ageing, development, behaviour, neurodegenerative
diseases
 Alzheimer’s Disease
 Post-mortem tissue compared with control tissue (age
matched, non demented)
 2D PAGE then N-terminal sequence analysis
 Found 37 proteins implicated – whose functions were
carbohydrate metabolism, lipid transport, stress
response and neurotransmission
 Some had already been identified in Alzheimer’s eg.
Superoxide dismutase
 Tau over-expression
 Linked to several neurodegenerative disorders including
Alzheimers, Pick’s disease etc
 Hyperphosphorylated Tau makes neurofibrillary tangles
 Made tau transgenic mice and compared them to
wildtype
 Showed 34 protein expression levels changed, to do with
energy metabolism, cytoskeletal integrity, signal
transduction and oxidative stress
 Cell death
 Made injury models based on DNA damage induced cell
death in cortical neurones
 Found changes in protein levels of proteins involved in
neurone growth rather than degeneration
 So some brain injuries are to do with abnormalities in
proteins which regulate growth and maintain neuronal
integrity
 Drug Discovery
 Find new targets
 Phage display is a protein binding screen
 ELISA shows drug interactions
 Reverse two hybrid shows disruptions of protein-protein
interactions