1. Proteomic Assessment of Thiol Modifications Victor Darley-Usmar, Ph.D. Center for Free Radical Biology, University of Alabama at Birmingham

2. Increased protein modification in cell signaling or oxidative stress ROS/RNS Modified proteins (altered function) nitrotyrosine thiol modification carbonyl formation

3. Proteomics is the study of a protein complement in response to a stimulus Potential for biomarkers Defining mechanisms Hypothesis Generation

4. Some Reactive Proteomes In Free Radical Biology Thiol Nitro Carbonyl Electrophile

5. 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.

6. –S - RSH –SH –S–S–R –S –SNO –SH NO, RNS –S–S– –SH HS– =O –SH ONOO - –SNO ROS =O –S–OH =O –SOH –SH =O –S–OH –SH Cooper et al. Trends Biochem. Sci. 2002 –SH –SR H O OH Sub-Classes of the Thiol Proteome

7. –S - RSH –SH –S–S–R –SNO –SH NO, RNS –S–S– –SH HS– ROS –SOH –SH –SR Modifications Discussed O –SX

8. –S - tag –S-tag signal Western blot/ Imaging –S X ROS/RNS signal Step 1: Are thiols modified at all?

9. Biotin as a tag N-(biotinoyl)-N'- (iodoacetyl)- ethylenediamine (BIAM) Advantages  Wide range of commercially synthesized tags available.  extremely sensitive when coupled with streptavidin/HRP  Can be used to pull down targets  Can be quantitative Less sensitive to local protein environment (c.f. antibodies)

10. Biotin as a tag N-(biotinoyl)-N'- (iodoacetyl)- ethylenediamine (BIAM) Disadvantage: Endogenous carboxylases 105K 75K bt-15d-PGJ 2 Biochem J 394:185-95 (2006) Mitochondria BIAM Cells BIAM

11. Cytochrome c: small (12,000 Da), water soluble, multiple surface lysine residues. Cytochrome c as an internal standard for protein and Biotin Biotin Tagging through Lysine:

12. Native Cytochrome c - 12360 Matrix Adduct - 12569 10000.01200014000160001800020000 Mass (m/z) 3 Biotins - 13374 4 Biotins - 13713 5 Biotins - 14052 2 Biotins - 13034 6 Biotins - 14391 7 Biotins - 14731 Apomyoglobin Standard 16952 8 Biotins - 15068 1 Biotin, 1 K - 12733 bt cyt.c 17588189 Biotin (pmol) 020406080100120140160180 0 500 1000 1500 2000 2500 3000 Band Density (Arbitrary Units) Biotin (pmol) Free Radic Biol Med. 40(3):459-68 (2006)

13. N-(biotinoyl)-N'- (iodoacetyl)- ethylenediamine (BIAM) Anal. Biochem. 283:214-221, 2000 Step 1:Prepare the sample and analyze by 1D-SDS-PAGE detect biotin (Western) Treatment lyse sample with BIAM at pH 8.0-8.5 Biochem J 379:359-366, 2004

14. Sypro Ruby stainbiotin blot Step 2: Application to a 2D-Proteomic Format (Rat Liver Mitochondria) Abundance Protein amt x dye binding Thiol Proteome Protein Amt x SH groups x reactivity

15. Biotin tag is more sensitive than the Sypro Stain -bt bt- protein biotin 0.3μg 0.01μg

16. abundance proteome is not the same as thiol proteome S-Bt tB-StB-S

17. –S - RSH –SH –S–S–R –SNO –SH NO, RNS –S–S– –SH HS– ROS –SOH –SH –SR O –SX

18. Diagonal electrophoresis for inter-protein disulfides hi low SH S S oxidative stress excise lane Identify proteins off of diagonal N-terminal Edman degradation sequencing Mass spectrometry Immunoblot and probe for candidate proteins S S S S Reduce S S Adapted from J Biol Chem. 2004 Oct 1;279(40):41352-60.

19. –S - GSH Cys –SH –S–S–R –SNO –SH NO, RNS –S–S– –SH HS– ROS –SOH –SH –SR O –SX

20. -biotin protein S-S- HS-X protein S- oxidizing environment -biotin S-X detection, purification, imaging, identification using avidin-based methodologies GSH GSH ester Cys X =

21. Protein Biotin

22. –S - RSH –SH –S–S–R –SNO –SH NO, RNS –S–S– –SH HS– ROS –SOH –SH –SR O –SX

23. Differences in structure due to PTM of SH group in Biology are subtle Surrounding amino residues will lead to epitope bias S N O RSOH RSO 2 H S-nitrosothiol Sulfenic Sulfinic

24. 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. protein SNO SOH -S-S Sulfenic acid S-nitrosothiol

25. Strategies Direct detection of the PTM. protein SOH -S-S protein SOH protein Does not react with thiol, sulfinic, sulfonic, disulfide, GSNO, Met Sulfoxide groups. Sulfenic Acid Dimedone

26. Strategies Differential chemical properties leading to specific insertion of a tag. protein SNO SOH -S-S BIOTIN SWITCH

27. protein SNO SOH -S-S Alkylation to block free S - Remove alkylating agent protein SNO SOH RS

28. ascorbate reduction proteinSNO proteinSOH R-S arsenite reduction Remove reagents Restore the SOH or SNO to S- proteinSBT proteinS-BT R-S TAG AFFINITY PURIFY and DETECT

29. Examples of RSNO/RSOH RSNO in endotoxin trtd macrophage Biotin Protein RSOH in peroxide (100  M) treated heart Biotin

30. pH Protein stained gel detect biotin Reactive Thiols Lyse and treat cells (BAEC) with BIAM DetaNONOate 2D-IEF How abundant are S-nitrosated Proteins?

31. 150 100 75 50 35 30 15 10 3 10 pH 3 10 pH Control-SH BlotAfter NO treatment-SH Blot Master map Total spots = 135 Matched =41 Matched Unmatched PNAS. 2004:101(1):384-9 70% thiols modified

32. 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%

33. The problem of false positives S-SR SX 30% SX PTM in a population of 20 proteins Convert Tag STag False Positive is 14% Block 93%effic.

34. The problem of false positives S-SRSX 5% SX PTM in a population of 20 proteins Convert Tag STag False Positive is 50%. Block 93%effic.

35. –S - RSH –SH –S–S–R –SNO –SH NO, RNS –S–S– –SH HS– ROS –SOH –SH –SR Detecting Specific Modifications O –SX

36. ADP ATP H+H+ H+H+ e-e- O2O2 H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ Future Directions; organelle specific P I + IBTP + –SH – S–TPP IgG

37. ControlEthanol Anti-IBTP Aldehyde dehydrogenase HSP70 2D SDS-PAGE followed by western blotting 1Pyruvate carboxylase129.6 19528/49 2Hsp7072.119428/74 3Hsp6060.9908/13 4Glutamate dehydrogenase56988/15 5Protein disulfide isomerase56.912310/9 6Mitochondrial aldehyde dehydrogenase5313512/15 7Acetyl-coenyzme A acyl transferase 241.8797/13 Mass (kDA) MOWSE score No. peptides matched/ unmatched 1 2 3 4 5 6 7 Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7.

38. 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

39. Current Lab Members 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 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

40. 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.