Transcript PROTEOME:

Genome of the week Enterococcus faecalis
• E. faecalis - urinary tract infections, bacteremia,
endocarditis.
• Organism sequenced is vancomycin resistant.
– Vancomycin is often last available antibiotic resistance to this drug often means no other
antibiotics will work.
• Major cause of nosocomial infections.
• Possible transfer of vanomycin resistance genes
to more serious pathogens is a major concern.
Genome of the week Enterococcus faecalis
• Over 25% of the E. faecalis genome consists of
foreign DNA.
– Phages, insertions sequences, transposons.
– Likely contributed to the acquisition of resistance to
multiple antibiotics.
• Over 35 PTS systems
– Responsible for transporting sugars into the cell.
– Most found in any sequenced genome, likely utilize
undigested sugars in the intestine.
The “omics” nomenclature…
Genomics
DNA (Gene)
Transcription
Transcriptomics
RNA
Translation
Functional
Genomics
Proteomics
PROTEIN
Enzymatic
reaction
Metabolomics
METABOLITE
Why study protein expression?
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
• 2D gel electrophoresis
– Method for separating and visualizing proteins
– Separation by charge and mass
• Mass spectrometry
– High throughput analysis and identification of
proteins.
– Fragmentation of proteins
– Analysis of peptides
• Book - pages 273-300.
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)
What determines the charge of a
protein?
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 the protein (to make
movement solely dependent on mass,
not shape) and eliminates charge.
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
Current Mass Spec Technologies
• Proteome profiling/separation
– 2D SDS PAGE - identify proteins
– 2-D LC/LC - high throughput analysis of lysates
(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)
– ITRAQ
Mass Spectrometry (MS)
• 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
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
2D - LC/MS
Methods for
protein
identification
Identifying proteins
• Trypsin - digest your protein
– Digests after R and K amino acids.
• Run peptide fragments on mass spec
• Digest the protein database “in silico”
• Compare mass spec data with theoretical
data.
• What must be true to identify your protein?
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)
How 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
• 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…
Advantages vs. Disadvantages
• Determination
of MW and aa.
Sequence
• Detection of
posttranslational
modifications
• High-throughput
capability
• High capital costs
• Requires sequence
databases for
analysis
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