Download 5-Proteome_Analysis_II(Ch 15&16)

Proteome and Gene
Expression Analysis
Chapter 15 & 16
The Goals
• Functional Genomics:
– To know when, where and how much
genes are expressed.
– To know when, where, what kind and how
much of each protein is present.
• Systems Biology:
– To understand the transcriptional and
translational regulation of RNA and
proteins in the cell.
Genes and Proteins
• First, we’ll talk about how to find out what genes are
being transcribed in the cell.
– This is often referred (somewhat misleadingly) to gene
“expression”.
• Second, we’ll look at measuring the levels of proteins
in the cell.
– The real “expression” of protein coding genes…
• Third, we’ll talk about how we process and analyze
the raw data using bioinformatics.
Review: Gene Arrays
• Put a bunch of different,
short single-stranded
DNA sequences at
predefined positions on
a substrate.
• Let the unknown
mixture of tagged DNA
or RNA molecules
hybridize to the DNAs.
• Measure the amount of
hybridized material.
Getting the Data
Getting Protein Expression
Data
• To be able to understand protein expression, we
need the concentrations of all proteins (the
“proteome”) in difference cell and tissue types under
varying conditions.
• Large scale identification of proteins is much more
limited than for RNA.
– Nothing really equivalent to RNA expression microarrays or
high-throughput sequencing exists yet.
• Relatively low-throughput technologies are all that we
have right now.
Measuring Protein Expression
• In order to measure all the types of
protein in a cell we must
– Extract the proteins
– Purify the proteins
– Identify the individual proteins
• How do we accomplish purification and
identification of proteins.
The Technologies:
Protein Expression
• Low-throughput
– 2D Gel Electrophoresis + Mass Spectrometry
– Liquid chromatograph + Mass Spectrometry
• Protein microarrays
– Limited in application at this point
– Can be used for things other than protein
expression like protein-protein interactions
Extracting the Proteins
• First, the proteins are extracted from the
cells using lysis.
– This involves a detergent that destroys the
membranes of the cell.
Separating the Proteins:
2D Gel Electrophoresis
• First step: pI/pH
– Proteins are introduced to a
gel with an imobilized pH
gradient.
– A charge is applied.
– Proteins migrate until the pH
causes them to lose their
charge (isoelectric point)
and then stop.
• Second step: mass
– First gel transferred to
second gel
– SDS (detergent) breaks
structure and charges the
proteins proportional to their
mass.
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Using the 2D Gel
• Staining makes the
spots containing the
individual (we hope)
proteins visible.
segmenting
– The gel is photographed.
– Protein level
(concentration) can be
estimated by image
processing.
• Individual, stained spots
can be cut out for
evaluation by Mass
Spectrometry.
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dust
“Two channel” 2D Gels
• Low signal-to-noise is a
problem with protein gels, as
it is with RNA expression
arrays.
– A similar trick of putting two
cell lysates (samples) on
one gel can help.
– Registration problems and
sample-dependent effects
are thereby minimized.
• However, 1-channel gels
allow comparing more than
two samples…
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Protein Identification Using
Mass Spectrometry
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Steps of Mass Spectrometry
• Digest:
– Sample (spot) is digested
with a proteolytic enzyme
• Spectrum:
– Peaks correspond to the
mass-charge ratio of protein
fragments
– These provide a fingerprint
• Identify:
– Compare fingerprint to
theoretical fingerprints
– Post-translational
modifications screw
things up.
Spectrum: Protein Fingerprint
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Tricks: Protein “chips”
• If you had an antibody to
every possible protein and
could put it on a chip, and
you could label the proteins
in your sample, you would
have something equivalent
to an RNA expression
microarray.
– Getting reliable antibodies is
difficult and expensive.
– Arrays with 500 to 2000
proteins are available
commercially; Clontech,
Eurogentec, Arrayit etc.
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Not part of this subject,
but cool…
Protein Arrays for Measuring
Protein-Protein Interactions
• You can synthesize
proteins from DNA
directly on a substrate.
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– Nano-well approach
– “Printing” approach:
DAPA (DNA to Protein
Array)
• These can be used for
measuring binding
between proteins, but
not for identification of
proteins.
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Next time:
Analyzing Gene and Protein Expression Data
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Gene expression clustering
Protein Expression Clustering