Download Chemical Approaches for Quantitative and Functional Proteomics

Chemical Approaches for
Quantitative and Functional
Proteomics
Carmen F. Drahl
Organic Supergroup Meeting
5 May 2004
“Central Dogma” Revisited
DNA
Genomics
RNA
Transcriptomics
Protein
Proteomics
Francis Crick, 1958
Mass Spec. Rev. 20, 1-57.
“Central Dogma” Revisited
Genomics
DNA
Transcriptional
regulation
RNA
Transcriptomics
Alternative splicing
mRNA editing
mRNA
Translational
regulation
Protein
Proteomics
These checkpoints are INVISIBLE
to many genomics techniques!
Protein/Protein
Post-translational modification
and small molecule
interactions
Proteolysis
Microb. Mol. Biol. Rev. 66, 39-63.
Why Not Genomics?
• Science and medicine need to derive
meaning from extensive genome data.
• One gene ≠ one protein
• Genome = static, Proteome = dynamic
• At the molecular level, function is most
closely associated with the biochemical
activities of proteins.
Function Æ Protein Activity
• Most drugs target proteins, yet disease profiling
is at present dominated by DNA microarrays.
Drug Name
Protein Target
2003 Global Sales
($bil)
Lipitor
HMG-CoA reductase
$10.3
Viagra
Phosphodiesterase-5
$1.7
Zyprexa
Serotonin and
dopamine receptors
$4.3
Comb. Chem. and H.T.S. 7, 1-9.
http://www.forbes.com
Difficulties Inherent in Proteomics
• Amplification: No
PCR equivalent exists
for protein.
• A paradigm shift from
identifying individual
players to
characterizing
interacting networks
and fluxes must occur
in biology.
Heat denature
dNTP's
Taq pol.
PCR reaction cycle
Proteomics Technologies Must..
• Display/ Separate/ Isolate
• Identify
and
• Quantify
large numbers of proteins
from complex mixtures.
Two Dimensional Electrophoresis
• First developed in
1975
• Detection by staining
– Coomassie blue
– Silver
– Sypro
• Mature proteomics
technology together
with mass spec.
J. Biol. Chem. 250, 4007-4021.
http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html
Applications for 2DE-MS
• Total Protein
– Global identification of “all” proteins in a sample
• Full proteome
• Fractionated tissue
• Can detect post-translational modifications
• Differential Expression
– Compare 2 or more samples and find differences in
their protein expression
• Overlaid on same gel (DIGE)
• Compare two or more gels
Anal. Chem. 75, 2891-2905.
Mass. Spec. Rev. 20, 1-57.
Drug Disc. Today 4, 55-62.
Address 2DE Technical Concerns
• Dynamic range of detection method
– Solution? multi-photon detection
• Overlapping proteins on gel
• Reproducibility
– Solution? Immobilized pH gradients
• Detection limits of gel for extremes of pI
and MW, low abundance proteins
Mass Spec. Rev. 20, 1-57.
Mass Spectrometry
• MS allows rapid, sensitive (1 pM or below) identification of
protein fragments in high complexity samples.
• MS may be used in tandem with separation techniques.
– 2DE
– LC
• The 2002 Nobel acknowledged the influence that improvements
in protein MS have had on the proteomics field.
Ionization Source
Mass Analyzer
Detector
MS Ionization for Proteomics
• Soft Ionizations
• Electrospray Ionization (ESI)
• Matrix-assisted laser desorption/ionization
(MALDI)
Nature 422, 198-207.
Mass Spectrometry
• MS Analysis of Proteins
– Peptide mass analysis/ mass fingerprinting
– Amino acid sequencing
y3
R1
y2
O
H
N
R3
H2N
O
b1
PNAS 83, 6233-6237.
y1
N
H
R2
b2
O
H
N
OH
O
b3
R4
Quantitative Proteomics
•
•
•
•
Isotope-coded Affinity Tag (ICAT)
Stable isotopes as internal standards
Gel-free
1 experiment Æ ID and relative abundance
O
NH
HN
H
H
H
N
S
O
Biotin affinity group
Nat. Biotech. 17, 994-999.
X
X
X
O
O
O
X
Isotopically labeled linker
X = H or X = D
X
X
X
X
H
N
I
O
Cys-specific reactive group
ICAT Applications
• Powerful method for global analysis of protein
abundance
– Different expression pattern following perturbation
• Nat. Biotech. 19, 946-951. (12-phorbol 13-myristate acid)
• Electrophoresis 23, 1591-8. (camptothecin)
• Systematic strategy for identifying candidate
drug targets
– ID proteins with binding selectivity for a drug
candidate over a structurally related analog
• Anal. Chem. 75, 2159-2165
ICAT Variants
CX3
X3C CX
H
N
O2 N
O
O
I
N
H
H
N
Br
OOC
COO
N
N
M
O
N
N
OOC
O
OMe
COO
Solid Phase ICAT
ECAT
O
N
H
O
H
H
H
N
S
O
Nat. Biotech. 20, 512-515.
Bioconj. Chem. 15, 3-6.
Bioconj. Chem. 15, 380-388.
I
NO2
NH
HN
N
H
O
VICAT
O
O
H
N
NO2
N
N
H
N
N O
Abs = 493 nm
Em = 503 nm
Disadvantages of ICAT
• Not every protein contains Cys
• Incomplete reactivity of tag with proteomes
• Complex mass spec. arising from
fragmentation of bulky tag, multiple Cys’s,
partial incorporation
• Protein level does not necessarily
correlate with function
Rapid Commun. Mass Spec. 17, 2380-2386.
Post-Translational Modifications
• Selected types
•Active site directed
– Phosphorylation protein regulation
– Acetylation
– Nitration
– Glycosylation
– Myristoylation
Nature 402, 373-376.
–Intrasteric
–Complementary to
allosteric
Phosphoproteomics Strategies
• Metabolic 32P radiolabeling
• Phospho-specific antibodies
• Immobilized metal affinity column
Mol. Cell. Proteom. 1, 781-790.
Chemical Phosphoproteomics
OPO3
Protein
X
Protein
X
S
S
• β-elimination method
• Phosphoramidate
method
steps
Protein
Biotin
X X
O
OPO3
X
OH
X
NH X
O P O
H
N
steps
Protein
X
Boc-HN
O
O
XX
X X
OH
steps
S
O
O
NH
O P O
H
N
Boc-HN
O
Mol. Cell. Proteom. 1, 781-790.
solid phase capture
XX
X X
OH
steps
O
O P O
H
N
Boc-HN
O
XX
X X
OH
Protein Microarrays
• Immobilized proteins on a solid support
– The folded conformation of proteins is
preserved.
• Examine interactions with tagged probes
– Small molecules
– Other proteins
• Develop molecular network profiles
Science 289, 1760-1763.
Proteomics 3, 2091-2100.
Activity Based Protein Profiling
• Abundance methods are indirect estimates of protein function.
• ABP Probes report on the functional state of enzymes in
complex proteomes.
– React with specific class(es) of enzyme directly in complex proteomes
– React in a manner correlating with catalytic activity
– Possess a tag for rapid, sensitive detection and isolation of products
"Warhead"
reactive group
Curr. Opin. Chem. Biol. 4, 663-668.
Mol. Cell Proteom. 1, 781-790.
Linker
specificity
Tag
Activity-Based Protein Profiling
• Directed ABPP
– Class specific irreversible inhibitors
• FP-Biotin
– Readily labels active serine hydrolases
– Labeled proteins ID’d by MALDI-MS/MS
O
F
H
N
P
O
O
HN
S
Biochem. 40, 4005
Mol. Cell Proteom. 1, 781-790.
O
NH
O
NH
Activity-Based Protein Profiling
• Other classes of cognate affinity labels
Tag
Tag
a
ax
Cl
R1
O
O
aax
O
O
Tag
Serine protease
O
H
N
Ub
O
O
N
H
Curr. Opin. Chem. Biol. 7, 296-303.
S
O
Et
Cysteine protease
(papain)
Cysteine protease
(Ub hydrolase)
Combinatorial/ Nondirected
ABPP Strategy
• Mild electrophile: sulfonate ester
• Labels enzymes from various mechanistic classes
– Aldehyde dehydrogenases
– Glutathione S-transferases
– phosphofructokinase
R=
H
N
O
R S O
O
Chem. Biol. 8, 81-95.
O
Tag
N
ABPP Applications
• The activity of a novel serine hydrolase
correlates with ovarian cancer cell invasiveness.
• FR182877 is a potent and selective inhibitor of
carboxylesterase-1
H
H3C
O
O
H3C
CH3
H
H
HO
H
PNAS 99, 10335-10340
ACIEE 42, 5480-5484.
O
H
O
O
H
H
H
(—)-FR182877
CH3
N N
N
9
Tag
Opportunities for Chemistry in the
Post-Genome Era
“The field of synthetic organic chemistry is
uniquely suited to furnish proteomics initiatives
with new concepts and tools that are
complementary and, in many cases, superior to
those in use by today’s researchers.”
Chemical methods will make significant
contributions to research efforts aimed at
recording variations in protein activity.
Curr. Opin. Chem. Biol. 4, 663-668.
Problem 1
Provide a mechanism for and the products of the transformation below.
CX3
X3C CX
H
N
O2 N
I
365 nm hν
O
O
N
H
N
H
O
OMe
O
Nature Biotech. 19, 512-515.
J. Chem. Technol. Biotechnol. 74, 835-851.
H2O
Provide a mechanism for and the products of the transformation below.
CX3
X3C CX
H
N
O
O N
N
H
O
O
H
O
OMe
I
O
O N
O
365 nm hν
OMe
H2O
O
O
N
H
Nitrobenzyl chromophore
small HOMO-LUMO gap
HO
Diradical representation
of excited state
H
N
O
N
O
O
H
N
OH
N
O
O centered radical abstracts H five atoms away
OMe
OMe
O
O
CX3
X3C CX
O
H
N
O
H O N
H
N
O N
H2N
O
O
OMe
O
Nature Biotech. 19, 512-515.
J. Chem. Technol. Biotechnol. 74, 835-851.
N
H
OMe
O
N
H
O
O
I
Problem 2
Cleavage at or near histidine is enhanced for peptides in
MS. Can you propose a fragmentation mechanism?
J. Mass Spec. 35, 1399-1406.
Problem 2 Hint
Cleavage at or near histidine is enhanced for peptides in MS.
Can you propose a fragmentation mechanism?
Below: “Normal” MS peptide fragmentation
H
R1
N
H
O
H
N
O
R3
N
H
R2
R1
N
H
O
N
H
H
N
O
O
J. Mass Spec. 35, 1399-1406.
R1
O
R1
R2
N
H
O
H
N
N
R2
H
N
O
R3
HH
O
R2
O
Problem 2
H
R
N
H
O
H
N
R2
N
H
O
R
O
N
H
O
H
N
N
H
O
N
N
H
H
N
O
N
H
R
N
H
N
Proton transfer
O
H
O
H
O N
R2
N
N
H
R2
J. Mass Spec. 35, 1399-1406.
H
N
O
N
H N
H H
O
N
N
H
R
R2
O