Transcript Human Origins

Chapter 8
Proteomics
Using high-throughput methods to
identify proteins and to understand their
function
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Contents
 Definition of proteomics
 Proteomics technologies
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2-D gel electrophoresis
Mass spectrometry
Protein chips
Yeast two-hybrid method
 Biochemical genomics
 Using proteomics to uncover transcriptional
networks
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What is proteomics?
 An organism’s proteome
 A catalog of all proteins
 Expressed throughout life
 Expressed under all conditions
 The goals of proteomics
 To catalog all proteins
 To understand their functions
 To understand how they interact with each
other
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The challenges of proteomics
 Splice variants create an enormous diversity of
proteins
 ~25,000 genes in humans give rise to 200,000
to 2,000,000 different proteins
 Splice variants may have very diverse functions
 Proteins expressed in an organism will vary
according to age, health, tissue, and
environmental stimuli
 Proteomics requires a broader range of
technologies than genomics
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Diversity of function in splice variants
 Example: the calcitonin gene
 Gene variant #1
 Protein: calcitonin
 Function: increases calcium uptake in bones
 Gene variant #2
 Protein: calcitonin gene-related polypeptide
 Function: causes blood vessels to dilate
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Posttranslational modifications
 Posttranslational modifications are defined as
any changes to the covalent bonds of a protein
after it has been fully translated.
 Proteolytic cleavage
 Fragmenting protein
 Addition of chemical groups to one or more
amino acids on the protein
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Chemical modifications
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Phosphorylation: activation and inactivation of enzymes
Acetylation: protein stability, used in histones
Methylation: regulation of gene expression
Acylation: membrane tethering, targeting
Glycosylation: cell–cell recognition, signaling
GPI anchor: membrane tethering
Hydroxyproline: protein stability, ligand interactions
Sulfation: protein–protein and ligand interactions
Disulfide-bond formation: protein stability
Deamidation: protein–protein and ligand interactions
Pyroglutamic acid: protein stability
Ubiquitination: destruction signal
Nitration of tyrosine: inflammation
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Practical applications
 Comparison of protein expression in diseased
and normal tissues
 Likely to reveal new drug targets
 Today ~500 drug targets
 Estimates of possible drug targets: 10,000–
20,000
 Protein expression signatures associated with
drug toxicity
 To make clinical trials more efficient
 To make drug treatments more effective
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Technologies for proteomics
 2-D gel electrophoresis (2-dimensional)
 Separates proteins in a mixture on the basis of their molecular
weight and charge
 Mass spectrometry
 Reveals identity of proteins based on computer software that can
uniquely identify individual proteins
 Protein chips
 A wide variety of identification methods
 structure, biochemical activity, and interactions with other
proteins
 Yeast two-hybrid method
 Determines how proteins interact with each other
 Biochemical genomics (Enzymatic)
 Screens gene products for biochemical activity
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2-D gel electrophoresis
 pH gradient along first
axis neutralizes
charged proteins at
different places
 pH constant on a
second axis where
proteins are separated
by weight
Basic
Low MW
 x–y position of proteins
on stained gel uniquely
identifies the proteins
Acidic
High MW
 Polyacrylamide gel
 Voltage across both axes
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Differential in gel electrophoresis
 Label protein samples
from control and
experimental tissues
 Fluorescent dye #1 for
control
 Fluorescent dye #2 for
experimental sample
 Mix protein samples
together
 Identify identical
proteins from different
samples by dye color
with
benzoic
acid
Cy3
without
benzoic
acid
Cy5
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Caveats associated with 2-D gels
 Poor performance of 2-D gels for the
following:
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Very large proteins
Very small proteins
Less abundant proteins
Membrane-bound proteins
 Presumably, the most promising drug targets
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Mass spectrometry
 Measures mass-tocharge ratio
 Components of mass
spectrometer
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Ion source
Mass analyzer
Ion detector
Data acquisition unit
A mass spectrometer
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Ion sources used for proteomics
 Proteomics requires
specialized ion sources
 Electrospray Ionization
(ESI)
 With capillary
electrophoresis and
liquid chromatography
 Matrix-assisted laser
desorption/ionization
(MALDI)
 Extracts ions from
sample surface
ESI
MALDI
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Mass analyzers used for proteomics
 Ion trap
Ion Trap
 Captures ions on the basis
of mass-to-charge ratio
 Often used with ESI
 Time of flight (TOF)
 Time for accelerated ion to
reach detector indicates
mass-to-charge ratio
 Frequently used with
MALDI
Time of Flight
Detector
 Also other possibilities
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A mass spectrum
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Identifying proteins with mass
spectrometry
 Preparation of protein sample
 Extraction from a 2-D gel
 Digestion by proteases — e.g., trypsin
 Mass spectrometer measures mass-charge ratio of
peptide fragments
 Identified peptides are compared with database
 Software used to generate theoretical peptide mass
fingerprint (PMF) for all proteins in database
 Match of experimental readout to database PMF allows
researchers to identify the protein
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Stable-isotope protein labeling
 Stable isotopes used to
label proteins under
different conditions
 Variety of labeling
methods
 Enzymatic
 Metabolic
 Via chemical reaction
 Relative abundance of
labeled and nonlabeled
proteins measured in
mass spectrum
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Data from a MALDI experiment
 Distributions of
individual proteins in a
slice of rat brain
 Tissue is coated with
UV-absorbing matrix
 MALDI ion source
with laser sampling
tissue every 180 mm
 Mass-spectrum peaks
reveal individual
proteins
 Image processing for
false-color images
Sections of rat brain imaged
by mass spectrometry
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Limitations of mass spectrometry
 Not very good at identifying minute quantities
of protein
 Trouble dealing with phosphorylated proteins
 Doesn’t provide concentrations of proteins
 Improved software eliminating human
mediated analysis is necessary for highthroughput projects
 Are only able to identify hundreds of proteins
in a single day
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Protein chips
 Thousands of proteins
analyzed simultaneously
 Wide variety of assays
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Antibody–antigen
Enzyme–substrate
Protein–small molecule
Protein–nucleic acid
Protein–protein
Protein–lipid
Yeast proteins detected
using antibodies
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Fabricating protein chips: physical array that can hold proteins, isolate them from
each other, and prevent them from becoming denatured
 Protein substrates:
minipads
 Polyacrylamide Polydimethylsiloxane
or
agarose gels
 Glass
 Nanowells
 Proteins deposited on
chip surface by robots
PDMS
UV
Quartz mask
Glass slide
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Protein attachment strategies
 Diffusion (pads)
 Protein suspended in
random orientation, but
presumably active
 Adsorption/Absorption
Diffusion
Adsorption/
Absorption
 Some proteins inactive
 Covalent attachment
Covalent
 Some proteins inactive
 Affinity
 Orientation of protein
precisely controlled
Affinity
antibodies
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Classes of capture molecules
Agent that interacts with molecules applied to the
chip to carry out some kind of assay
 Different capture molecules
must be used to study
different interactions
 Examples
 Antibodies (or antigens) for
detection
 Proteins for protein-protein
interaction
 Enzyme-substrate for
biochemical function
 analytical microarrays and
functional protein chips
Antigen–
antibody
Protein–
protein
Aptamers:
short peptides
Enzyme–
substrate
Receptor–
ligand
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Reading out results
 Fluorescence
 Most common method
 Fluorescent probe or tag
 Can be read out using standard nucleic acid microarray
technology
 Surface-enhanced laser desorption/ionization (SELDI)
 Laser ionizes proteins captured by chip
 Mass spectrometer analyzes peptide fragments
 Atomic-force microscopy
 Detects changes in chip surface due to captured
proteins
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Difficulties in designing protein chips
 Unique process is necessary for constructing each
probe element
 Challenging to produce and purify each protein on
chip
 Proteins can be hydrophobic or hydrophilic
 Difficult to design a chip that can detect both
 Protein’s function may be dependent on
posttranslational modification or an interaction with
another biological molecule
 Challenging and constantly improving with new
technological advancements
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Regulation of transcription
UE
TATA box
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Yeast two-hybrid method
 Goal: Determine how proteins interact with each other
 Method
 Use yeast transcription factors
 Gene expression requires the following:
 A DNA-binding domain
 An activation domain
 A basic transcription apparatus
 Attach protein1 to DNA-binding domain (bait)
 Attach protein2 to activation domain (prey)
 Reporter gene expressed only if protein1 and protein2
interact with each other
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A schematic of the yeast two-hybrid method
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Results from a yeast two-hybrid experiment
 Goal: To characterize protein–protein
interactions among 6,144 yeast ORFs
 5,345 were successfully cloned into yeast as
both bait and prey
 Identity of ORFs determined by DNA
sequencing in hybrid yeast
 692 protein–protein interaction pairs
 Interactions involved 817 ORFs
 interactome on a genome-wide scale
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Caveats associated with the yeast
two-hybrid method
 There is evidence that other methods may be
more sensitive (protein chip)
 Some inaccuracy reported when compared
against known protein–protein interactions
 False positives
 False negatives
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Subcellular localization of the yeast
proteome
 Complete genome sequences allow each ORF
to be precisely tagged with a reporter molecule
 Tagged ORF proteins indicate subcellular
localization
 Useful for the following:
 Correlating to regulatory modules
 Verifying data on protein–protein interactions
 Annotating genome sequence
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Attaching a GFP tag to an ORF
Marker gene: HIS free medium
GFP
PCR product
HIS3MX6
Homologous
recombination
Chromosome
Fusion protein
ORF1
NH2
protein
ORF2
GFP COOH
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Location of proteins revealed
 75% of yeast proteome
localized
 > 40% of proteins in
cytoplasm
 67% of proteins were
previously un-localized
 Localizations correlate
with transcriptional
modules
cytoplasm
nucleus
A protein localized
to the nucleus
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Pregenomics biochemical assays
 Methods used to find genes responsible for
specific biochemical activity before the
inception of genomics
 Laboriously purify responsible protein
 Often expensive and time consuming
 Expression cloning
 Introduce cDNA pools into cells
 Look for biochemical activity in those cells
 Caveat: Often difficult to detect biochemical
activity in cell’s biochemical “background”
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Biochemical genomics
 Genome of an organism is already known
 Approach
 Construct plasmids for all ORFs
 Attach ORFs to sequence that will facilitate
purification
 Transform cells
 Isolate ORF products
 Test for biochemical activity
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Biochemical genomics in yeast
 6,144 ORF yeast strains made
 ORFs fused to glutathione S-transferase (GST) for
purification purposes
 Biochemical assay revealed three new biochemical
reactions associated with yeast ORFs
tRNA ligase
pre-tRNA
Ligated tRNA
tRNA halves
Abc1
35
64
Used as screening pools
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Microfluidics
 Proteomics requires
greater automation
 Microfluidics: a “lab on
a chip”
 Microvalves and
pumps allow control of
nanoliter amounts
 Can control
biochemical reactions
A microfluidics chip
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Microfluidics in action
loading
mixing
compartmentalization
purging
500 mm
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Finding transcription-factor targets
 All yeast transcription
factors were used to
make yeast strains
 Use chromatin
immunoprecipitation to
select factors attached to
promoter regions on
DNA (ChIP)
 DNA fragments used on
microarray to identify
transcription-factor
targets (ChIP on chip)
antibodies
Genomic DNA
Over 106 TFs of 141 are
active
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Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Network motifs for transcriptional regulation
Autoregulation
Multicomponent loop
Multi-input motif
TF
Motif
Regulator chain
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Studying proteins with
posttranslational modifications
 Example: tyrosine phosphorylation
 Traditional method
 Radioactive labeling with 32P followed by gel
electrophoresis or chromatography
 Problems using mass spectrometry are being
overcome to allow high-throughput analysis
 Better purification techniques
 Mass spectrometers more capable of detecting
phosphorylated peptides
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HUPO
 Human Proteome Organization (HUPO) was
established in 2002
 Mission
 Consolidate proteomics organizations in
different countries into single worldwide body
 Scientific and educational programs to help
spread proteomics knowledge and technology
 Coordination of public proteomics initiatives
 Examples of current initiatives
 Human Liver Proteome Project
 Human Plasma Proteome Project
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Future prospects
 The next decade may see the complete
deciphering of the proteome of yeast
 More initiatives, like the Human Liver
Proteome Project, are underway
 Better understanding of disease
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Summary I
 Goals of proteomics
 Identify and ascribe function to proteins under
all biologically plausible conditions
 Proteomics methods
 2-D gel electrophoresis for separating proteins on the
basis of charge and molecular weight
 Mass spectrometry for identifying proteins by
measuring the mass-to-charge ratio of their ionized
peptide fragments
 Protein chips to identify proteins, to detect protein–
protein interactions, to perform biochemical assays, and
to study drug–target interactions
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Summary II
 Proteomics methods (continued)
 Yeast two-hybrid method for studying protein–protein
interactions
 Biochemical genomics for high-throughput assays
 Some accomplishments of proteomics
 Example: yeast
 Yeast two-hybrid method reveals interactome
 Transcriptional regulatory networks deduced
 Biochemical genomics uncovers new ORF
functions
 Subcellular localization of proteins
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Summary III
 Future prospects
 Better technology for studying posttranslational
modifications
 ~10 years for completion of yeast proteome?
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