Transcript SH - sfrbm
Proteomic Assessment of Thiol Modifications Victor Darley-Usmar, Ph.D. Center for Free Radical Biology, University of Alabama at Birmingham Increased protein modification in cell signaling or oxidative stress ROS/RNS nitrotyrosine thiol modification carbonyl formation Modified proteins (altered function) Proteomics is the study of a protein complement in response to a stimulus Potential for biomarkers Defining mechanisms Hypothesis Generation Some Reactive Proteomes In Free Radical Biology Thiol Nitro Carbonyl Electrophile Role of thiols in protein function and cell signaling “redox signaling” Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine. Mitchell DA, Marletta MA Nat Chem Biol. 2005 Aug;1(3):154-8. Epub 2005 Jul 10. Sub-Classes of the Thiol Proteome –S–S–R –SNO –SH –SH RSH –SNO –S- HS– ROS ROS –SH O –S –SOH H OH –S –SH –SR Cooper et al. Trends Biochem. Sci. 2002 –S–OH –S–OH –SH –SH =O –SH =O ONOO- –S- –SH =O =O NO, RNS –S–S– Modifications Discussed –S–S–R –SNO –SH –SH RSH NO, RNS –S- –S–S– –SH HS– –SROS O –SR –SH –SX –SOH –SX –SH Step 1: Are thiols modified at all? tag –S- –S-tag Western blot/ ROS/RNS –SX signal Imaging signal Biotin as a tag Advantages Wide range of commercially synthesized tags available. extremely sensitive when coupled with streptavidin/HRP N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine (BIAM) Can be used to pull down targets Can be quantitative Less sensitive to local protein environment (c.f. antibodies) Biotin as a tag BIAM BIAM bt-15d-PGJ2 Disadvantage: Endogenous carboxylases 105K 75K N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine (BIAM) Biochem J 394:185-95 (2006) Mitochondria Cells Cytochrome c as an internal standard for protein and Biotin Cytochrome c: small (12,000 Da), water soluble, multiple surface lysine residues. Biotin Tagging through Lysine: Band Density (Arbitrary Units) Native Cytochrome c - 12360 Matrix Adduct - 12569 Biotin (pmol) 9 3000 18 88 175 bt cyt.c 2500 2000 1500 1000 500 0 0 20 40 60 80 100 120 140 160 180 Biotin (pmol) 3 Biotins - 13374 4 Biotins - 13713 5 Biotins - 14052 2 Biotins - 13034 6 Biotins - 14391 7 Biotins - 14731 Apomyoglobin Standard 8 Biotins - 15068 16952 1 Biotin, 1 K 12733 10000.0 12000 14000 Mass (m/z) Free Radic Biol Med. 40(3):459-68 (2006) 16000 18000 20000 Step 1:Prepare the sample and analyze by 1D-SDS-PAGE Treatment lyse sample with BIAM at pH 8.0-8.5 N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine (BIAM) Anal. Biochem. 283:214-221, 2000 Biochem J 379:359-366, 2004 detect biotin (Western) Step 2: Application to a 2D-Proteomic Format (Rat Liver Mitochondria) Sypro Ruby stain biotin blot Thiol Proteome Abundance Protein amt x dye binding Protein Amt x SH groups x reactivity Biotin tag is more sensitive than the Sypro Stain -bt biotin protein 0.3μg 0.01μg abundance proteome is not the same as thiol proteome S-Bt S-Bt –S–S–R –SNO –SH –SH RSH NO, RNS –S- –S–S– –SH HS– –SROS O –SR –SH –SX –SOH –SX –SH Diagonal electrophoresis for inter-protein disulfides S S hi SH oxidative stress excise lane SH Reduce low S Identify proteins off of diagonal S N-terminal Edman degradation sequencing Mass spectrometry Immunoblot and probe for candidate proteins Adapted from S S S S J Biol Chem. 2004 Oct 1;279(40):41352-60. –S–S–R –SNO –SH –SH GSH Cys NO, RNS –S- –S–S– –SH HS– –SROS O –SR –SH –SX –SOH –SX –SH protein S- HS-X -biotin oxidizing environment GSH X = GSH ester Cys protein S-S-X-biotin detection, purification, imaging, identification using avidin-based methodologies Protein Biotin –S–S–R –SNO –SH –SH RSH NO, RNS –S- –S–S– –SH HS– –SROS O –SR –SH –SX –SOH –SX –SH Differences in structure due to PTM of SH group in Biology are subtle Sulfenic S-nitrosothiol S N Sulfinic RSOH O RSO2H Surrounding amino residues will lead to epitope bias SNO -S protein SOH S-nitrosothiol Sulfenic acid Strategies Direct detection of the PTM. Antibody: epitope too small and not structurally distinct. Mass Spectrometry: Sensitivity often not adequate Differential chemical properties leading to specific insertion of a tag. -S protein SOH Sulfenic Acid Strategies Direct detection of the PTM. Dimedone protein protein SOH Does not react with thiol, sulfinic, sulfonic, disulfide, GSNO, Met Sulfoxide groups. SNO -S protein SOH Strategies Differential chemical properties leading to specific insertion of a tag. BIOTIN SWITCH SNO -S protein SOH Alkylation to block free S- Remove alkylating agent SNO RS protein SOH Restore the SOH or SNO to S- R-S protein SOH arsenite reduction R-S protein SNO Remove reagents ascorbate reduction TAG R-S protein S-BT R-S protein SBT AFFINITY PURIFY and DETECT Examples of RSNO/RSOH Biotin Protein RSNO in endotoxin trtd macrophage Biotin RSOH in peroxide (100 mM) treated heart How abundant are S-nitrosated Proteins? DetaNONOate Lyse and treat cells (BAEC) with BIAM 2D-IEF detect biotin Reactive Thiols Protein stained gel pH Control-SH Blot 3 pH After NO treatment-SH Blot 10 3 pH 10 150 100 75 50 35 30 15 10 Master map Total spots = 135 Matched =41 Matched Unmatched PNAS. 2004:101(1):384-9 70% thiols modified Measure RSNO and thiols by direct non-proteomics technique. RSNO 11.2 ± 0.07pmol/mg protein Protein Thiol approx 40-80 nmol/mg protein 0.014-0.028% The problem of false positives 30% SX PTM in a population of 20 proteins Block 93%effic. S- SR SX STag False Positive is 14% Convert Tag The problem of false positives 5% SX PTM in a population of 20 proteins S- SR SX STag Block 93%effic. Convert False Positive is 50%. Tag Detecting Specific Modifications –S–S–R –SNO –SH –SH RSH NO, RNS –S- –S–S– –SH HS– –SROS O –SR –SH –SX –SOH –SX –SH Future Directions; organelle specific –SH H+ H+ + H + H + H+ H H+ + H+ H H+ eO2 + H ATP ADP S–TPP – IgG P IBTP+ H+ + I 2D SDS-PAGE followed by western blotting Control Anti-IBTP Ethanol HSP70 1 2 3 5 1 2 3 4 5 6 7 6 4 Aldehyde dehydrogenase 7 Mass MOWSE No. peptides matched/ unmatched (kDA) score Pyruvate carboxylase 129.6 195 28/49 Hsp70 72.1 194 28/74 Hsp60 60.9 90 8/13 Glutamate dehydrogenase 56 98 8/15 Protein disulfide isomerase 56.9 123 10/9 Mitochondrial aldehyde dehydrogenase 53 135 12/15 Acetyl-coenyzme A acyl transferase 2 41.8 79 7/13 . Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7 Challenges Matching the proteome with tag pattern Developing internal standard for gel and blot Secondary reactions may also lead to thiol Modification Thiol proteomes are composed of discreet low abundance proteins Current Lab Members Elena Ulasova Joo-Yeun Oh Jessica Gutierrez Brian Dranka Balu Chacko Ashlee Preston Jeff Dubuisson Former Members Nobuo Watanabe Jaroslaw Zmijewski Claire Le Goffe Niroshini Giles Anna-Liisa Levonen Sruti Shiva Collaborators Aimee Landar Anne Diers Yeun Su Choo Karina Ricart Michelle Johnson Stephen Barnes Paul Brookes Dale Dickinson Jason Morrow Lewis Pannell Shannon Bailey Neil Hogg Scott Ballinger Philip Eaton Bruce King Selected References for Thiol Proteomics • • • • • • • • • • • • • • • • Eaton, P. (2006) Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures. Free Radic Biol Med 40, 1889-1899 Good overview of the various methods available for measuring thiol redox status in a proteomics context and the principles involved. Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M. and King, S. B. (2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug Chem 16, 1624-16028. Example of the strategies to develop a thiol tag that can be applied to proteomics. Landar, A., Oh, J. Y., Giles, N. M., Isom, A., Kirk, M., Barnes, S. and Darley-Usmar, V. M. (2006) A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress. Free Radic Biol Med 40, 459-468 Method for the quantitative measurement of biotin tags in proteomics gel formats. Patton, W. F. (2002) Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 771, 3-31 Broad overview of the various approaches to assessing post-translational modification of proteomes. Gao, C., Guo, H., Wei, J., Mi, Z., Wai, P. Y. and Kuo, P. C. (2005) Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages. Nitric Oxide 12, 121-126. An application of the biotin switch method as applied to S-nitrosothiols showing endogenous protein S-nitrosation. Gladwin, M. T., Wang, X. and Hogg, N. (2006) Methodological vexation about thiol oxidation versus S-nitrosation -a commentary on "An ascorbate-dependent artifact that interferes with the interpretation of the biotin-switch assay". Free Radic Biol Med 41, 557-561 Discussion of the problem of false positives in biotin switch methods. Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y. and Liebler, D. C. (2006) Cytosolic and nuclear protein targets of thiol-reactive electrophiles. Chem Res Toxicol 19, 20-29 Use of mass spectrometry proteomics analysis to define the electrophile responsive proteome in cells. Levonen, A. L., Landar, A., Ramachandran, A., Ceaser, E. K., Dickinson, D. A., Zanoni, G., Morrow, J. D. and Darley-Usmar, V. M. (2004) Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem J 378, 373-382 An example of the candidate protein approach using different tagging approaches to identify modification of a cell signaling molecule.