Download 2016-10-12 Jurgen Chemical Proteomics

Chemical Proteomics
Jürgen Eirich, 2016-10-12
Outline
Definitions and general considerations
The three parts of a chemical proteomics project:
1: The small molecule
2: The protein sample
3: Identification and quantification
Your lab project
Literature / Reviews
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Chemical Proteomics...
… is a multidisciplinary science
spanning molecular and cell biology, biochemistry, pharmacology,
analytical and organic chemistry, physics, informatics,…
… aims to study how small molecules (“chemicals”) of synthetic or
natural origin bind to proteins and modulate their function.
… can be applied in drug target discovery or to identify
small-molecule probes as research tools to study protein function.
… often relies on current state-of-the-art in protein mass spectrometry
(MS) as analytical strategy.
DOI: 10.1007/978-1-61779-364-6
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Definitions
A small molecule is a low molecular weight organic
compound (< 900 Daltons) that may help regulate a
biological process. Most drugs are small molecules.
A drug is any substance other than food, that when
consumed causes a physiological change in the body.
A chemical compound (or just compound if used in the
context of chemistry) is an entity consisting of two or more
atoms, at least two from different elements, which associate
via chemical bonds.
wikipedia.org – to encourage further reading...
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A probe
A chemical probe is a selective small-molecule modulator of a protein’s function
that allows the user to ask mechanistic and phenotypic questions about its
molecular target in biochemical, cell-based or animal studies.
potency and selectivity
target-dependent action (in cell-based or a cell-free context)
structure and/or well-characterized derivative known
utility as a probe to address the molecular target
available to the academic community with no restrictions on use
(in an ideal case…)
DOIs: 10.1038/nchembio.1867, 10.1038/nchembio.296, 10.1038/nature09504
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Different purposes and requirements for
chemical probes and drugs.
• may have undefined mechanism of action
(MoA)
• defined MoA is essential
• selectivity
• intelectual property (IP) restrictions;
limited availability
• compound itself and activity data (freely)
available
• must be bio-available
• drug-like properties not necessarily required
• high bar for physicochemical and
pharmaceutic properties
• structurally related inactive and structurally
unrelated active compounds
DOI:10.1038/nchembio.1867
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Typical three parts of a project
Typical three parts of a project (and this lecture):
1: selection, design and/or synthesis of the compound(s)
2: exposure to the protein samples
3: identification and quantification of target(s)
DOI:10.1038/nchembio.216
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Typical three parts of a project: summary
1: selection, design and/or synthesis of the compound(s)
discovery of small molecule protein targets
generation of specific chemical probes
compound repurposing
affinity capture (beads, micorarrays)
tagged probes (biotin, bio-orthogonal “click” handles (alkyne, azide, …))
unmodified/label-free compounds
enzyme classes vs. single proteins (drug target)
protein kinases, dehydrogenase enzymes, adenine-binding proteins, lipid
electrophiles
(known) onco-protein/variant
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Typical three parts of a project: summary
2: exposure to the protein samples
(complex, physiological) environment:
lysate, intact cells, model organism, clinical material
(rather than on isolated individual proteins)
enrichment of target proteins in the presence of competing free test compound
Include cross-linking to study protein–drug interactions, or ligand modulation of
protein–protein interactions
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Typical three parts of a project: summary
3: identification and quantification of target(s)
targeted vs. untargeted/unbiased
antibodies vs. fluorescent dyes vs. MS
quantitative MS with isobaric mass tags or metabolic labeling to determine the
potency for a target
find target protein(s)
off-targets (as the cause of side effects)
biomarkers for treatment
research tool compound vs. drug candidate
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Typical three parts of a project
Examples from current literature
to explain/highlight certain aspects
in one of the typical three parts of a
chemical proteomics project/paper.
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1: The compound
discovery of small molecule (off) targets
generation of specific chemical probes
compound repurposing
DOI: 10.1146/annurev-biochem-060713-035708
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Aspirin-Dependent Protein Modification
in Living Cells
Aspirin
covalent inhibition of cyclooxygenase enzymes
through non-enzymatic acetylation of key
serine residues
identification of 120 proteins, 112 of which had
not been previously reported to be acetylated
by aspirin
DOI: 10.1021/ja408322b
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1: The compound
affinity capture (beads, micorarrays)
tagged probes (biotin, click handles (alkyne, azide, …))
unmodified compounds
DOI: 10.1146/annurev-biochem-060713-035708
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Analysis of glycans using a bio-orthogonal
chemical reporter strategy.
attachment of a bio-orthogonal functional group (an azide -N3) to chemically
modified precursors of monosaccharides (sugars)
incorporated into glycans by living cells after uptake
detection by bio-orthogonal ligation and adding a compound that carries a
readily detectable tag
DOIs: 10.1038/nprot.2007.422, 10.1038/nbt1004-1244
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1: The compound
enzyme classes vs. single proteins (drug target)
protein kinases, dehydrogenase enzymes, adenine-binding proteins, lipid
electrophiles
(known) onco-protein/variant
DOI: 10.1146/annurev-biochem-060713-035708
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Kinobeads
Strategy for assessing
inhibitor selectivity in
proteomes:
kinase
native
Kinobeads use immobilized,
broad-spectrum kinase ligands
to enrich active kinases from
complex proteomes.
DOI: 10.1146/annurev-biochem-060713-035708
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Kinobeads
compound concentration
protein displacement
protein compound affinity
DOIs: 10.1038/nbt1328 & 10.1021/pr5012608
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Targets of Imatinib
Western blot analysis of proteins captured
on kinobeads:
Imatinib treatment of lysate (top panel) and
cells in culture (second panel) similarly
reduces the amount of target protein DDR1
captured on kinobeads.
phosphorylation-specific
Y703P-KIT
antibody (third panel) and a general KIT
antibody (bottom panel) show that only the
phosphorylated species are affected by
Imatinib.
Discoidin Domain Receptor family, member 1
receptor Tyrosine KInase
DOIs: 10.1038/nbt1328 & 10.1021/pr5012608
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The other way around
A high-throughput, multiplexed assay for
superfamily-wide profiling of enzyme activity
Engineered, immobilized enzymes, but unmodifyed compounds,
Fluoresence read out...
DOI: 10.1038/nchembio.1578
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2: The protein sample
environment:
lysate, intact cells, model organism, clinical material
(rather on isolated individual proteins)
enrichment of target proteins in the presence of competing, free test compound
Include cross-linking to study protein–drug interactions, or ligand modulation of
protein–protein interactions
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In vivo imaging
C. elegans embryos are labeled with the sugar-derived probe Ac4GalNAz (OAcetylated N-AZidoGalactosamine) and reacted with DIFO-488 (green) for
detection via fluorescent microscopy
incubated again with Ac4GalNAz to metabolically label a second, newer population
of glycans
reacted with DIFO-568 (red) to distinguish the old from the new populations
DOI: 10.1021/cb900254y
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In vivo imaging
DIC: differential interference contrast, 488 and 568 fluorescence channels
Larval stages L1 (top panels), L2 (middle panels), or adult (bottom panels)
C. Elegans metabolically labeled with azide-sugars were reacted with DIFO-488
and DIFO-568 in different developmental stages. Yellow indicates co-localization.
DOI: 10.1021/cb900254y
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Competition assay in theory…
inhibitor administered to a biological system
(cell, animal model)
proteomes are isolated and
treated with a probe suitable for the targets of interest
selective labeling of (still) active (uninhibited) enzymes.
DOI: 10.1146/annurev-biochem-060713-035708
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… and practice
By competition with a enzyme class
specific probe the selectivity of inhibitors
for a target protein is measured
DOI: 10.1021/jm400898x
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3: Identification and quantification
targeted vs. unbiased
antibody-based vs. fluorescence vs. MS
quantitative MS with isobaric mass tags or metabolic labeling to determine the
potencies for a large number of targets in a single analysis.
Find
target protein(s)
off-targets (as the cause of side effects)
biomarkers for treatment
binding sites
DOI: 10.1146/annurev-biochem-060713-035708
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Isotope-coded taggs and linkers
ICAT (Isotope Coded Affinity Tag) reagent:
mass difference of 8 Da at
a charge state of two
three elements:
an affinity tag (biotin), to isolate labeled peptides;
a linker with stable isotopes (X);
a reactive group specific towards thiol groups of cysteines.
The reagent exists in two forms, heavy and light where X either represents
either deuterium or hydrogen leading to a mass shift.
DOI: 10.1038/13690
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Binding site identification
MS/MS sequencing shows the binding of
the aspirin probe to Lys46 of histone H1.4
Locations of aspirinmodified residues in the
protein GAPDH.
Numbers of the
aspirin-modified
amino acid
residues.
DOI: 10.1038/srep07896
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Protein stabilization by ligand binding
When proteins in their native conformation are subjected to denaturing
conditions, they lose higher-order structures, leading to the unfolded state.
Protein unfolding, or denaturation, can be deterred by the presence of a bound
ligand (e.g. buffer component, drug, …).
These changes are the bases for different assays that identify ligands or other
conditions that increase the stability of a protein.
DOI: 10.1056/NEJMcibr1308868
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Protein stabilization by ligand binding
protein (+/- ligand)
isolated
or
in a cell
denaturing conditions
isolation of (native) proteins
identification
DOI: 10.1056/NEJMcibr1308868
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Denaturing conditions
(a) Drug Affinity Responsive Target Stability (DARTS):
Ligand bound proteins are more resistant to
proteolysis in the presence of denaturant. Non-binding proteins are hydrolyzed.
Proteolysis resistant proteins can be analyzed by MS.
DOIs: 10.1016/j.cbpa.2012.12.022, 10.1039/C6MD00045B
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Denaturing conditions
DOIs: 10.1016/j.cbpa.2012.12.022, 10.1039/C6MD00045B
(b) Stability of Proteins from Rates of OXidation (SPROX):
Ligand bound proteins are more resistant to oxidant, thus
requiring higher amounts of denaturant to generate the same degree of oxidation
compared to non-binders.
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CEllular Thermal Shift Assay
Western blot quantification
DOIs: 10.1038/nprot.2014.138, 10.4155/fmc.15.50, 10.1146/annurev-pharmtox-010715-103715
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MS TPP
Thermal Proteome Profiling (TPP):
a) Temperature Range (TR) experiment
b) IsoThermal-Dose-Response (ITDR)
Encoding temperatures or compound
concentrations as TMT channels
DOIs: 10.1038/nprot.2015.101, 10.1038/nmeth.3590
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Hands on - Lab course
The three parts of your lab project:
1: selection, design and/or synthesis and
purchase of the probe
Azide-tagged probe for click mediated pull down
2: exposure to the protein samples
In cell lysate
3: identification and quantification of target(s)
via MS
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Literature: Review Articles
Chemical Proteomics - Methods and Protocols, Editors: G. Drewes, M. Bantscheff
DOI: 10.1007/978-1-61779-364-6
Target profiling of small molecules by chemical proteomics, U. Rix, G. Superti-Furga
DOI:10.1038/nchembio.216
Target Identification for Small Bioactive Molecules: Finding the Needle in the Haystack,
S. Ziegler, et al., DOI: 10.1002/anie.201208749
Enzyme Inhibitor Discovery by Activity-Based Protein Profiling, M. Niphakis, B. Cravatt
DOI: 10.1146/annurev-biochem-060713-035708
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ABPP: Activity-Based Protein Profiling
probe
reporter
Curr Opin Chem Biol. 2012 (1-2): p 227-233.
Probe design
Reactive group:
mechanism-based inhibitors,
(protein-reactive) natural products
general electrophilic moieties
photoreactive chemotypes
Binding group many vary in
non-covalent interactions
size
hydrophobicity
Tags:
Dye, Biotin, Azide, Alkyne, …
Annu Rev Biochem (2008) 77: 383 - 414
Photo cross linkers
Aryl azides
Alkyl/Aryl diazirines
Benzophenones
Affinity-Based Probe (AfBP)
Top Curr Chem (2012) 324: 85-113