PROTEOMICS
LECTURE
The “omics” nomenclature…
Genomics
DNA (Gene)
Transcription
Transcriptomics
RNA
Translation
Functional
Genomics
Proteomics
PROTEIN
Enzymatic
reaction
Metabolomics
METABOLITE
A few definitions…
Gen
Transcript
Prote
Metabol
Gen
Prote
~ome = Sequence of a
complete set of
~omics =
Analysis of the
Genes
Transcripts
Proteins
Metabolites
Genome
Proteome
Why study protein expression?
(The steps of gene expression control)
(Gygi et al., Mol. Celll. Biol., 1990, p.1720-1730)
Nucleus
Cytosol
Inactive mRNA
RNA
Degradation
control
DNA
Primary
RNA
transcript
mRNA
mRNA
Translation control
Transcriptional
control
RNA
Processing
control
RNA
Transport
control
protein
Modified
protein
Post-translational
control
Applications of Proteomics
• Mining: identification of proteins (catalog
the proteins)
• Protein-expression profile: identification of
proteins in a particular state of the
organism
• Protein-network mapping: protein
interactions in living systems
• Mapping of protein modifications: how and
where proteins are modified.
Proteins classes for Analysis
•
•
•
•
•
•
•
Membrane
Soluble proteins
Nuclear
Chromosome-associated
Phosphorylated
Glycosylated
Complexes
SEPARATION
IDENTIFICATION
General
flow for
proteomics
analysis
Current Proteomics Technologies
• Proteome profiling/separation
– 2D SDS PAGE (two-dimensional sodium
dodecylsulphate polyacrylamide gel electrophoresis)
– 2-D LC/LC
(LC = Liquid Chromatography)
– 2-D LC/MS
(MS= Mass spectrometry)
• Protein identification
– Peptide mass fingerprint
– Tandem Mass Spectrometry (MS/MS)
• Quantative proteomics
- ICAT (isotope-coded affinity tag)
2D-SDS
PAGE gel
1) Sample loading
3)Place the strip gel
in the focusing tray
2) Remove the cover
sheet from the IEF gel
4) Place the strip on the
top of the SDS-PAGE gel
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
(pH gradient makes the net charge 0)
Isoelectric point (pI)
• Separation by charge:
4
Stable pH gradient
5
Low pH:
Protein is
positively
charged
6
7
8
9
10
High pH:
protein is
negatively
charged
At the isolectric
point the protein
has no net
charge and
therefore no
longer migrates
in the electric
field.
2D-SDS PAGE gel
The first dimension
(separation by isoelectric focusing)
- gel with an immobilised pH gradient
- electric current causes charged
proteins to move until it reaches the
isoelectric point
(pH gradient makes the net charge 0)
The second dimension
(separation by mass)
-pH gel strip is loaded onto a SDS gel
-SDS denatures and linearises the
protein (to make movement solely
dependent on mass, not shape)
2D-SDS PAGE gel
2D-gel technique example
Advantages vs. Disadvantages
• Good resolution
of proteins
• Detection of
posttranslational
modifications
• Not for
hydrophobic
proteins
• Limited by pH
range
• Not easy for low
abundant proteins
• Analysis and
quantification are
difficult
2D - LC/LC
Study protein
complexes
without gel
electrophoresis
Complex mixture is
simplified prior to
MS/MS by 2D LC
(trypsin)
Peptides all bind
to cation
exchange column
Successive elution
with increasing salt
gradients separates
peptides by charge
Peptides are
separated by
hydrophobicity on
reverse phase
column
Reverse Phase column
Polypeptides enter the column in the mobile phase…
…the hydrophobic “foot” of the polypeptides adsorb to the
hydrophobic (non polar) surface of the reverse-phase
material (stationary phase) where they remain until…
…the organic modifier concentration rises to critical
concentration and desorbs the polypeptides
2D - LC/MS
Methods for
protein
identification
Mass Spectrometry (MS) Stages
• Introduce sample to the instrument
• Generate ions in the gas phase
• Separate ions on the basis of differences in m/z
with a mass analyzer
• Detect ions
How the protein sequencing works?
• Use Tandem MS: two mass
analyzer in series with a collision
cell in between
• Collision cell: a region where
the ions collide with a gas (He,
Ne, Ar) resulting in fragmentation
of the ion
• Fragmentation of the peptides in
the collision cell occur in a
predictable fashion, mainly at the
peptide bonds (also
phosphoester bonds)
• The resulting daughter ions have
masses that are consistent with
known molecular weights of
dipeptides, tripeptides,
tetrapeptides…
Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc…
Tandem Mass Spectrometry
(trypsin)
Isolates individual peptide fragments for
2nd mass spec – can obtain peptide
sequence
Compare peptide sequence
with protein databases
Advantages vs. Disadvantages
• Determination
of MW and aa.
Sequence
• Detection of
posttranslational
modifications
• High-throughput
capability
• High capital costs
• Requires sequence
databases for
analysis
Protein identification by Peptide
Mass fingerprint
• Use MS to measure the masses of
proteolytic peptide fragments.
• Identification is done by matching the
measured peptide masses to
corresponding peptide masses from
protein or nucleotide sequence databases.
Mass spectometry (MS)
(trypsin)
eg. MALDI-TOF
Mass spectrometry – method of separating
molecules based on mass/charge ratio
Compare peptide m/z
with protein databases
Protein Identification by MS
Library
Spot removed
from gel
Artificial
spectra built
Fragmented
using trypsin
Spectrum of
fragments
generated
MATCH
Artificially
trypsinated
Database of
sequences
(i.e. SwissProt)
ISOTOPE-CODED AFFINITY TAG
(ICAT): a quantitative method
• Label protein samples with heavy and light
reagent
• Reagent contains affinity tag and heavy or light
isotopes
Chemically reactive group: forms a
covalent bond to the protein or peptide
Isotope-labeled linker: heavy or light,
depending on which isotope is used
Affinity tag: enables the protein or
peptide bearing an ICAT to be isolated by
affinity chromatography in a single step
Example of an ICAT Reagent
Biotin Affinity tag:
Binds tightly to
streptavidin-agarose
resin
Reactive group: Thiolreactive group will bind to Cys
O
Linker: Heavy version will
have deuteriums at *
Light version will have
hydrogens at *
NH
NH
H
N
*
S
O
*
O
O
*
O
*
H
N
I
O
How ICAT works?
Affinity isolation
on streptavidin
beads
Lyse &
Label
Quantification
MS
NH2-EACDPLR-COOH
Light
100
Identification
MS/MS
100
MIX
Heavy
Proteolysis
(eg trypsin)
0
550
570
m/z
590
0
200
400
m/z
600
Advantages vs. Disadvantages
• Estimates relative
protein levels between
samples with a
reasonable level of
accuracy (within 10%)
• Can be used on
complex mixtures of
proteins
• Cys-specific label
reduces sample
complexity
• Peptides can be
sequenced directly if
tandem MS-MS is used
• Yield and non specificity
• Slight chromatography
differences
• Expensive
• Tag fragmentation
• Meaning of relative
quantification information
• No presence of cysteine
residues or not accessible
by ICAT reagent