The Research Journal of Kendall Dutcher 2005 2005 - 2007 2007 Primary Primary Mentor: Mentor: Dr. Dr. Douglas Douglas F. F. Barofsky Barofsky Dept. Dept. of of Chemistry, Chemistry, Oregon
Oregon State State University, University, Corvallis, Corvallis, OR OR 97331 97331 Research Topics I. Selenoprotein W: A Search for Selenosulfide Bonding (2005-06) II. Severity of Damage Inflicted on
Intramolecular Disulfide Bonds as a Result of UV Radiation (200607) Mass Spectrometry Mass spectrometer: measures masses of individual molecules that have been converted to ions Mass spectrometry (MS) is a useful analytical technique that is used to identify unknown compounds quantify materials elucidate structure and chemical properties of molecules Mass Spectrometry
Basic components of a mass spectrometer Mass Spectrometry Molecules must be charged (via ionization) to be analyzed via MS Ionization Methods MALDI (matrix-assisted laser desorption/ionization) impact of high-energy photons on sample imbedded in a solid organic matrix
ESI (electrospray ionization) formation of charged liquid droplets from which ions are desolved or desorbed Mass Spectrometry MALDI-TOF/TOF and target plate Mass Spectrometry HPLC-ESI-MS/MS and syringe
Mass Spectrometry MALDI-MS/MS (tandem) is ideal for peptide analysis MS/MS MS #1 Fragmentation Chamber MS #2 Sorting Molecules
Breaking Molecules Sorting Pieces Basic tandem MS setup Research Topic I Selenoprotein W: A Search for Selenosulfide Bonding What is Selenoprotein W? Selenium
essential trace nutrient Incorporated as selenocysteine (SeC) into proteins At least 10 animal Periodic Table of Elements selenoproteins Ex: glutathione peroxidase and thioredoxin reductase. What is Selenoprotein W? Nutritional deficiency of selenium decreases selenoprotein concentrations and leads to pathologic conditions.
HUMANS LIVESTOCK Keshan Disease White Muscle Disease Endemic cardiomyopathy of children and women of childbearing age reported in selenium-deficient areas such as China, New Zealand, and Finland Leads to calcification of
muscle; as well as decrease oxidant defense, thyroid hormone metabolism, and defense against viral infections Selenoproteins presumably mediate these biologic effects What is Selenoprotein W? Non-native SeW peptide sequence Selenoprotein W (SeW) first purified from rat skeletal muscle 1992 Cloned cDNA sequences revealed one in-frame codon for SeC per polypeptide sequence
Precise function of SeW unknown, thought to play role in selenium or calcium metabolism White Muscle Disease (WMD) Radio-labeled selenium, incorporated into SeW, was shown to be reduced in WMD lambs Sarcoplasmic reticulum in muscle of WMD animals loses its ability to sequester calcium, resulting in calcification of both skeletal and cardiac muscle QuickTime and a
TIFF (Uncompressed) decompressor are needed to see this picture. View of the cardiac white muscle of a lamb that had White Muscle Disease Although selenium is normally incorporated into membranes (as selenocysteine), it is unclear how selenium is involved in their maintenance and/or function Characterization of SeW SeW binds to glutathione and may play some role in redox mechanisms SeW may also be a glutathione-dependent in vivo
antioxidant This activity and the presence of selenocysteine in SeW suggest that there may be intermolecular selenosulfide bonds in the N-terminal region SeW (E. coli, non-native) peptide sequence and suggested disulfide linkage Hypothesis (a) Native form of SeW will have a seleniumsulfur (selenosulfide) bond in N-terminal region of protein where a disulfide bond has been found to exist in non-native SeW Method Isolated native SeW obtained from pigs was analyzed (prepared by Bauman in a previous study, believed to exist in various
stages of purification) The partially purified proteins were to be further isolated using SDS-PAGE electrophoresis (~12% acrylamide) Bands corresponding to approximately 10kDa were excised and digested using pepsin. (-) Method Method Reduced or non-existant bond
+2mu Oxidized Process of analysis Results Peptide CGACGYKPKY m/z = 1062.4 when reduced m/z = 1060.4 when oxidized
Database analysis was able to support the idea that these ion peaks were indeed the CGAC sequence. These observations suggested that there was a disulfide bond present in the non-reduced form of SeW Identifiable peaks of the peptide CGACGYKPKY Results Mass spectrum of oxidized non-native SeW
Results Mass spectrum of reduced non-native SeW Results Partially Purified SeW SDS PAGE Electrophoresis Band Excision TCEP Reduction Pepsin Digestion MALD MS
No SeW No SeW No SeW Conclusions 1) There was either no SeW is in the samples or its concentration was too low to be detected via MALDI MS (<10-100fmol), 2) The protein had degraded over time to a point that it was no longer identifiable, possibly due to proteolysis or chemical hydrolysis, or 3) The procedure for isolating and analyzing the protein may be inappropriate.
References
Keshan Disease Research Group of the Chinese Academy of Medical Sciences (1979) Observations on effect of sodium selenite in prevention of Keshan disease. Chinese Med. J. 92: 471-476. Hill, K. E.; Dasouki, M.; Phillips, J. A., III; Burk, R. F (1996) Human selenoprotein P gene maps to 5q31. Genomics 36: 550551. Vendeland, S; Beilstein, A; Chiareiy, C; Barofsky, E; Whanger, P.D. (1993) Purification and Properties of Selenoprotein W from Rat Muscle. J. Biol. Chem. 268:17103-7. Sies, H.; Lester, L (Eds.) (2002) Methods in Enzymol. Protein Sensors and Reactive Oxygen Species Part A: Selenoproteins and Thiorodoxin, 347. Tripp, M.J.; Whanger, P.D.; Schmitz, J.A. (1993) Calsium Uptake and ATPase Activity of Sarcoplasmic Reticulum Vesicles Isolated from Control and Selenium Deficient Lambs. J. Trace Elem. Electrolytes Health Dis. 7:75-82. Whanger, P.D. (2000) Selenoprotein W: a review. Cell. Mol. Life Sci. 57:1846-52 Whanger, P.D. Selenoprotein W (2002) in Sies, H. and Lester, L (2002) See Above. Beilstein M.A., S.C. Vendeland, E. Barofsky, O.N. Jensen, P.D. Whanger (1996) Selenoprotein W of Rat Muscle Binds Glutathione and an Unknown Small Molecular Weight Moiety, J. Inor. Biochem., 61, 117-124. Jeong D.W., T.S. Kim, Y. W. Chung, B. J. Lee, I. Y. Kim (2002) Selenoprotein W is a Glutathione-dependent Antioxidant in Vivo, FEBS Lett., 517, 225-228. Bauman A.T., D.A. Malencik, D.F. Barofsky, E. Barofsky, S.R. Anderson, P.D. Whanger . (2004) Selective production of rat
mutant selenoprotein W with and without bound glutathione. Biochem Biophys Res Commun. Jan 9; 313 (2):308-313. Bauman A.T. (Jan. 2004) Phosphorylation Studies of SeW. PhD thesis, Oregon State University, Corvallis, OR. Vendeland S.C., MA. Beilstein, J.Y. Yeh, W.L. Ream, P.D. Whanger (1995) Rat skeletal muscle selenoprotein W: cDNA clone and mRna modulation by dietary selenium. Proc. Natl. Sci. USA 92, 8749-8753. Gu Q.P., M.A. Beilstein, S.C. Vendeland, W. Lugade, L.W. Ream, P.D. Whanger (1997) Conserved features of selenosysteine insertion sequence (SECIS) elements in seleoprotein Research Topic II Severity of Damage Inflicted on Intramolecular Disulfide Bonds as a Result of UV Radiation Background Sun Alert: Limiting sun exposure, wearing protective clothing, and using
sunscreens may reduce the risks of skin aging, skin cancer and other harmful effects of the sun. -- back label of this bottle of sunscreen Background/Theory High energy radiation itself may be directly responsible for only part of the damage Damage caused by intermediate species could be far more significant because of the chemical reactions that follow Intermediate species:
excited atoms and molecules ions radicals and secondary electrons produced in very high abundance (>10 4 by a 1MeV projectile) Molecules within irradiated cells interact with secondary electrons and other radiation-produced species causing mutagenic, genotoxic and other potentially damaging DNA and protein lesions
Background Secondary or slow electrons Thought to be harmful to biomolecules i.e. DNA, cause mutations Little is known about effects on proteins Consider cataracts: UV production of oxygen free radical (superoxide) disrupts DNA Slow electrons may be similar may disrupt disulfide bonds and cause incorrect shuffling Disrupting disulfide bonds lead to hazy effect in lens, a.k.a. cataracts Hypothesis UV radiation (in the form of secondary electrons) will cause the intramolecular
disulfide bonds to break in proteins, like Glutathione (GSSG) Method GSH and GSSG (100uM, 1xPBS) Exposure to UVB and UVC at a range of intensities Range of UV intensity: 0-32 MEDs; five sets: 0, 4, 8, 16, and 32* Both protein batches treated with NEM (Nethylmaleimide** and internal standard (Glu-Val-Phe) * 1 MED = 200 J/m2, enough to irritate the skin ** Protects free thiols of any GSH and affords a stable adduct for LC separation Method Free GSH or GSSG
UVB or UVC Radiation + Control (no radiation) DNTB Colorimetric Assay NEM & Internal Standard Addition Quantification of GSH HPLC-MS Identity Confirmation of Proteins Qualitative Confirmation of Decrease in GSSG Method
Analysis and quantification DTNB colorimetric assay Statagene Statalinker 1800 SpectraMax microplate reader (@412nm) Colorimetric assay process Method HPLCLCQ-MS/MS* * Thermo Finnigan LCQ Deca XP Plus ion trap mass spectrometer coupled to Thermo Finnigan Surveyor HPLC system, Thermo
Hypercarb column (100mm x 2.1mm) Results GSH Molarities at Varying UV Intensities GSH @ 254nm GSH @ 365nm Molarity (uM) 26.0 24.0 254nm: -9.51.3% 365nm: -8.82.1%
22.0 20.0 0 100 200 300 400 500
600 700 800 UV Intensity (mJ/cm2) DTNB colorimetric assay results for GSH @ 254nm and 365nm Results GSH Molarities at Varying UV Intensities GSSG @ 254nm Molarity (uM)
2.0 1.5 1.0 0.5 Increase of ~1uM 0.0 0 200 400
600 UV Intensity (mJ/cm2) DTNB colorimetric assay results for GSSG @ 254nm 800 Results Next step: separate UV treated proteins via HPLC and analyze with MS NEM-GSH Problems:
Co-elution Possible contamination Time Theoretical elution times of GSSG, NEM-GSH and Glu-Val-Phe (Harwood et al, 2006) Results Each species (GSH-NEM, GSSG) was fragmented and monitored
GSH and GSSG gave m/z parent ions consistent with what had been observed in previous literature The protonated GSSG (m/z 613) and GSHNEM conjugate (m/z 433) gave fragment ions (m/z 484 and 304 respectively) that represented a neutral loss of 129 Da, but intensities were not stong enough to be conclusive Theoretical Selective Reaction Monitoring mass/charge values for GSSG, GSH-NEM, Glu-Val-Phe Conclusions If the coelution and contamination problems can be overcome, results could be forthcoming In vivo studies would be the next step References
ejkov, J., tpek, S., Crkovsk, J., Ardan, T., Pltenk, ejka, ., Midelfart, A. (2004). UV rays, the prooxidant/antioxidant imbalance in the cornea and oxidative eye damage. Physiol. Res.(Minireview). 53, 1-10. LaVerne, J.A., Pimblott, S.M. (1955). Electron energy loss distributions in solid, dry DNA. Radiat. Res.141 (2), 208-215. Cobut, V., Frongillo, Y., Patau, J.P., Goulet, T., Fraser, M.-J., Jay-gerin, J.-P. (1998). Monte Carolo simulation of fast electron and proton tracks in liquid water I. Physical and physiocochemical aspects. Radiat. Phys. Chem. 51, 229-243.
von Sonntag, C. (1987). The chemical basis for radiation biology. Ed. Taylor and Francis, London, UK. Fuciarelli A.F., Zimbrick, J.D., Eds. (1995). Radiation damage in DNA : structure/function relationships at early times. Battelle Press, Columbus. Sanche, L. (2002). Nanoscopic aspects of radiobiological damage fragmentation induced by secondary low-energy electrons. Mass Spectrom. Rev. 21, 349-369. ICRU (1979). International Commission on Radiation Units and Measurements. ICRU Rept. ICRU, Washington, DC.
Yamamoto, O. (1976). Aging Carcinogenesis and Radiation Biology. Plenum Press, New York, NY. Ward, J.F. (1977). Advances in Radiation Biology, v. 5, Academic Press, New York, NY. Gafken, P.R. (2000) Characterization of UV-crosslinked protein-nucleic acid interfaces by MALDI MS and ESI MS/MS. Unpublished. Harwood, T.D.; Kettle, A.J; Winterbourn, C.C. (2006) Production of glutathione sultonamide and dehydroglutathione from GSH by
myeloperoxidase-derived oxidants and detection using a novel LC-MS/MS method. Biochem. J. 399, 161-168 Pearson, P.G.; Howard, W.N.; Nelson, S.D. (1990), Screening strategy for the detection of derivatized glutathione conjugates by tandem mass spectrometry Acknowledgments Dr. Douglas F. Barofsky SeW Project
Dr. Larry Curtis Dr. Andrew Karplus Ms. Sarah Sowell Dr. Andrew Bauman Ms. Lilo Barofsky Mr. Rick Scheri Dr. Ben Figard Dr. Kevin Marley GSH Project Dr. Samuel Bennett Mr. Brian Arbogast Dr. Max Deinzer
Financial Contributors B.A. Gilman Foundation NIEHS URISC (OSU) E.R. Jackman Foundation Florence Garden Club And special thanks those who sparked my initial interest in biochemistry: Ms. Judy Butler and Dr. Phillip Whanger
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