Proteomic Assessment of Thiol Modifications Victor Darley-Usmar, Ph.D.

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

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

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

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

5. Role of thiols in protein function and cell signaling “redox signaling”

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

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

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

9. N-(biotinoyl)-N'- (iodoacetyl)-ethylenediamine (BIAM) Biotin as a tag • 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. N-(biotinoyl)-N'- (iodoacetyl)-ethylenediamine (BIAM) Biotin as a tag Disadvantage: Endogenous carboxylases BIAM BIAM bt-15d-PGJ2 105K 75K Biochem J 394:185-95 (2006) Cells Mitochondria

11. 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:

12. 9 18 88 175 Biotin (pmol) 3000 bt cyt.c 2500 2000 Band Density (Arbitrary Units) 1500 1000 500 0 0 20 40 60 80 100 120 140 160 180 Biotin (pmol) Native Cytochrome c - 12360 Matrix Adduct - 12569 3 Biotins - 13374 4 Biotins - 13713 5 Biotins - 14052 2 Biotins - 13034 6 Biotins - 14391 7 Biotins - 14731 Apomyoglobin Standard 16952 1 Biotin, 1 K - 12733 8 Biotins - 15068 10000.0 12000 14000 16000 18000 20000 Mass (m/z) Free Radic Biol Med. 40(3):459-68 (2006)

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

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

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

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

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

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

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

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

21. Biotin Protein

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

23. Differences in structure due to PTM of SH group in Biology are subtle Sulfinic Sulfenic S-nitrosothiol S RSOH Surrounding amino residues will lead to epitope bias RSO2H O N

24. 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.

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

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

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

28. Restore the SOH or SNO to S- R-S R-S protein SOH protein SNO ascorbate reduction arsenite reduction Remove reagents TAG R-S R-S protein S-BT protein SBT AFFINITY PURIFY and DETECT

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

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

31. pH pH 3 10 3 10 Master map Total spots = 135 Matched =41 Matched Unmatched PNAS. 2004:101(1):384-9 Control-SH Blot After NO treatment-SH Blot 150 100 75 50 35 30 15 10 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. 93%effic. Block The problem of false positives 30% SX PTM in a population of 20 proteins S- SR SX STag False Positive is 14% Convert Tag

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

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

36. + P I IBTP+ –SH –S–TPP IgG Future Directions; organelle specific H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ e- O2 H+ ATP ADP H+

37. 1 2 3 4 5 6 7 2D SDS-PAGE followed by western blotting Control Ethanol Anti-IBTP HSP70 Aldehyde dehydrogenase No. peptides matched/ unmatched Mass (kDA) MOWSE 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.

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. Collaborators 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

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.