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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