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Identification of Protein Free Radicals Using Mass Spectrometry Leesa J. Deterding

Identification of Protein Free Radicals Using Mass Spectrometry Leesa J. Deterding Laboratory of Structural Biology National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, NC. Outline. Advantages of Mass Spectrometry MS Tools

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Identification of Protein Free Radicals Using Mass Spectrometry Leesa J. Deterding

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  1. Identification of Protein Free Radicals Using Mass Spectrometry Leesa J. Deterding Laboratory of Structural Biology National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, NC

  2. Outline • Advantages of Mass Spectrometry • MS Tools • Applications of MS to Protein Radicals • Horse Myoglobin • Sperm Myoglobin • Hemoglobin • Limitations of MS… and how to alleviate them • Summary Deterding, 2005

  3. Advantages of Mass Spectrometry • High sensitivity • Low sample requirements (10 ug – 1 mg) • Universal detector • Inhomogeneous mixtures • Intact, functional proteins • glycosylated, no truncations • biologically relevant Deterding, 2005

  4. Mass Spectrometric Tools • Electrospray Ionization: • • normal ESI at 3-5 ul/min • • nanoflow at 200 nL/min – low sample consumption • • compatible with separations • • compatible with MS/MS • MALDI: • • tolerant of “MS dirty” samples • • not compatible with flow separations • • compatible with MS/MS • typically, more sensitive than ESI • Tandem Mass Spectrometry: MS/MS Sequencing • Separation Techniques: • • HPLC, SDS-PAGE, and affinity Deterding, 2005

  5. + + + + To MS + + + + + + + + + + + + + ESI Needle + + Electrospray Ionization Process Sample 3-5 ul/min 200 nl/min Desolvated Ions Droplet Formation Sampling Region Deterding, 2005

  6. 100 0 600 1000 1200 1600 800 1400 100 0 10000 11000 12000 13000 14000 ESI Mass Spectrum of Horse Cytochrome C +16 773.5 +17 728.0 +15 825.0 Raw MS Data +18 687.6 +14 883.9 +13 951.7 +19 651.5 +12 1031.0 +11 1124.6 +10 1237.0 m/z 12360.5 Deconvoluted Data Mass Deterding, 2005

  7. + + + + + + + + + + MALDI Ionization Process Sample Target Laser Flight Tube + + + + + + Detector c + + + + c + + + c + + + + + + + + + + Gas Phase Ions Deterding, 2005

  8. 5600 9200 12800 16400 20000 2000 MALDI Mass Spectrum of Horse Cytochrome C +2 6183.1 +1 12360.3 +3 4120.4 +4 2452.6 Mass (m/z) Deterding, 2005

  9. 100 0 100 0 12480 12340 12200 12500 12350 12200 Comparison of Mass Resolution Capabilities for Intact Protein 12360.5 ESI/MS 12360.3 MALDI/MS Deterding, 2005

  10. Tandem Mass Spectrometry (MS/MS) • Structural Analysis Tool • mass spectrum of a mass spectrum • compatible with ESI and MALDI • biomolecules fall apart into building blocks • Information gained • amino acid sequence • site(s) of modification collision cell MS-2 MS-1 Analyte Mixture parent ion selection fragment ion detection Deterding, 2005

  11. 100 0 100 0 100 0 200 400 600 800 1000 1200 1400 1600 1800 MS/MS of m/z 587.3+3 from Mixture 742.5 MS Scan 880.5 692.4 1043.9 587.3 1112.6 330.7 587.3 Ion Selection For MS/MS 136.1 MS/MS Scan 269.1 583.3 639.9 696.4 745.9 569.3 784.4 464.6 m/z Deterding, 2005

  12. b2 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 D-R-V-Y-I-H-P-F-H-L-L-V-Y-S y8 y3 y2 100 b10 b12 b9 ITyr y2 b6 (M+H)+ b8 b5 b13 y3 y8 b4 b7 b2 0 200 400 600 800 1000 1200 1400 1600 MS/MS of m/z 587.3+3 b11 Deterding, 2005

  13. Protein Radicals • Protein radicals traditionally studied by Electron Spin Resonance (ESR) Spectroscopy • Radicals are typically short-lived (msec-sec) • “Spin Trapping” – Protein radicals react with a “spin trap” molecule, thereby, making the radical more stable and more long-lived. • MNP: 2-methyl-2-nitrosopropane • DMPO: 5,5-dimethyl-1-pyrroline N-oxide • Reaction Conditions: • Protein + Spin Trap + H2O2 Protein Radical Adduct Deterding, 2005

  14. Formation of Protein Radical Adducts Deterding, 2005

  15. Data Dependent Acquisition MS and MS/MS info LC/MS Only Preferred MS Approach to Protein Radical Identification Sample Cleanup MS of Intact Protein Adducts ESI/MS MALDI/MS Enzymatic Digestion of Protein Adducts LC/MS Analyses of Protein Adduct Digest LC/MS/MS of Targeted Masses Deterding, 2005

  16. ESR Analysis 50 mM Mb DMPO-Spin Trapping on Horse Myoglobin Deterding, 2005

  17. X-Ray Structure of Horse Myoglobin Deterding, 2005

  18. HH Myo + DMPO + H2O2 +1 DMPO 17061.6 16951.6 100 0 16500 17000 17500 Deconvoluted ESI/MS of Horse Myoglobin 16952.0 HH Myo 100 0 16500 17000 17500 Detweiler, C.D., Deterding, L.J., Tomer, K.B., Chignell, C.F., Germolec, D., and Mason, R.P.: Free Rad. Biol. Med., 33: 364-369, 2002. Deterding, 2005

  19. LC/ESI/MS Analysis of Tryptic Digest of Radical Adducts 153.58 100 155.65 157.82 160.37 161.63 162.79 175.83 95.66 98.58 177.43 112.92 81.07 80.00 79.66 213.19 0 Time 50.00 100.00 150.00 200.00 Deterding, 2005

  20. LC/ESI/MS Analysis of Tryptic Digest of Radical Adducts 112.9 min 153.5 min 167.9 min (T16+DMPO+2H)2+ Deterding, 2005

  21. b3 b5 b7 b9 b10 b11 b12 b13 b14 b15 103Y-L-E-F-I-S-D-A-I-I-H-V-L-H-S-K118 y14 y13 y11 y10 y9 y7 y6 y5 y4 y3 DMPO + (M+H) 2+ (M+2H) X20 100 -DMPO b y y y 14 y Tyr-DMPO y 9 b b b 13 b 11 14 y 6 y y 7 y 9 15 5 b b b 5 4 3 b 7 y b 10 12 3 13 10 11 0 200 800 1400 2000 Mass LC/MS/MS of T16 of Horse Myoglobin Detweiler, C.D., Lardinois, O.M., Deterding, L.J., Ortiz de Montellano, P., Tomer, K.B., and Mason, R.P.: Free Rad. Biol. Med., 38: 969-976, 2005. Deterding, 2005

  22. Oxidatively Damaged Hemoglobin Lane A: Hb (10 mM) Lane B: Hb + DMPO Lane C: Hb + H2O2 Lane D: Hb + H2O2 + DMPO A B C D A B C D kDa 188 98 62 catalase 49 trimer 38 dimer 28 17 monomer 14 6 3 Western Analysis Silver Stain Deterding, 2005

  23. Alpha Chain 15128.5 Beta Chain 15869.0 100 0 mass 14600 14800 15000 15200 15400 15600 15800 16000 16200 16400 100 0 mass 14600 14800 15000 15200 15400 15600 15800 16000 16200 16400 ESI/MS of Hemoglobin Samples Hb Control Hb + DMPO + H2O2 + 1 DMPO 15239.8 + 1 DMPO 15980.6 15869.1 15128.8 Deterding, 2005

  24. Cys93 His20 Tyr42 Tyr24 Tyr24 Tyr42 His20 X-Ray Structure of Hemoglobin Tetramer Alpha chain – cyan Beta chain – purple Heme - red Deterding, L.J., Ramirez, D.C., Dubin, J.R., Mason, R.P., and Tomer, K.B.: J. Biol. Chem.,279: 11600-11607, 2004. Deterding, 2005

  25. b2 b4 b5 17V-G-A-H-A-G-E-Y-G-A-E-A-L-E-R31 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 y14 y13 x2 x2 DMPO 100 100 b2 b4 b5 b7 b8 b6 0 0 100 100 200 200 300 300 400 400 500 500 600 600 700 700 800 800 900 900 1000 1000 1100 1100 1200 1200 1300 1300 1400 1400 1500 1500 1600 1600 Mass Mass 17V-G-A-H-A-G-E-Y-G-A-E-A-L-E-R31 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 y14 y13 DMPO LC/MS/MS of T4 of Alpha Chain of Hemoglobin (M+H)+ -DMPO -H2O * y12 * * * (M+2H)2+ * y7 * y12 –DMPO -H2O y4 y1 b4 y8 y5 (M+H)+ -DMPO -H2O y10 y12 y11 b5 (M+2H)2+ His-DMPO b4 y7 y8 y4 y12 –DMPO -H2O y1 y5 Deterding, 2005

  26. Cys93 His20 Tyr42 Tyr24 Tyr24 Tyr42 His20 X-Ray Structure of Hemoglobin Tetramer Alpha chain – cyan Beta chain – purple Heme - red Deterding, L.J., Ramirez, D.C., Dubin, J.R., Mason, R.P., and Tomer, K.B.: J. Biol. Chem.,279: 11600-11607, 2004. Deterding, 2005

  27. Limitations of Mass Spectrometry • Suppression Effects • Additives • Complex mixtures • Negatively charged modifications • Stringent “Additive” Requirements • Unacceptable additives: • Glycerol Preservatives • Detergents Imidazoles • Salts • Universal Detector • Not selective, i.e. “MS sees everything” • Modifications of unknown mass Deterding, 2005

  28. How can we alleviate the limitations? • Separation Techniques • Online: LC/MS • Offline: microdialysis, HPLC, FPLC • 2D-LC/MS • Affinity Techniques • Immobilized Antibodies • Selective Enrichment Techniques • Immobilized metal affinity chromatography (IMAC) • Biotin-Streptavidin Binding (Kd = 1013-1015 M-1) Deterding, 2005

  29. Complementary Detection of Protein Radicals Anti-DMPO Ab No No No ug Yes Mass Spec Yes Yes Yes ug No ESR No Yes No mg Yes Protein Id Amino Acid Id Specific residue Sensitivity Selectivity Deterding, 2005

  30. Summary • Peptide mapping and MS is powerful technique for detection and determination of protein radicals • MS, ESR, and antibody detection are complementary • Major radical site on myoglobin (horse and sperm whale) is at Tyr-103 • Multiple radical sites are formed on hemoglobin: • Alpha Chain: Tyr-42, Tyr-24, and His-20 • Beta Chain: Cys-93 Deterding, 2005

  31. Acknowledgements Mass Spectrometry Workgroup Laboratory of Structural Biology Ken Tomer Free Radical Metabolism Workgroup Laboratory of Pharmacology and Chemistry Ronald Mason Charles Detweiler Dario Ramirez Deterding, 2005

  32. 100 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 Mass LC/MS/MS of T10-11 of Beta Chain of Hemoglobin b2 b3 b4 b5 b10 b17 83G-T-F-A-T-L-S-E-L-H-C-D-K-L-H-V-D-P-E-N-F-R104 y20 y19 y18 y17 y16 y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 y5 y3 y1 (M+H)+ y5 DMPO -DMPO -H2S y16-DMPO-H2S y20 y8 y16 y18 y6 y14 y17 y19 y10 y7 y11 y9 b3 y12 y13 b5 y1 Deterding, 2005

  33. b2 b3 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 41T-Y-F-P-H-F-D-L-S-H-G-S-A-Q-V-K56 y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 y15 y14 y13 DMPO x2 100 b7 b14 -H20 y7 b5 y6 y8 y14 y11 y10 y13 y12 y9 b12 y2 b3 y1 b2 y15 0 200 400 600 800 1000 1200 1400 1600 1800 Mass LC/MS/MS of T6 of Alpha Chain of Hemoglobin (M+H)+ Deterding, 2005

  34. 16600 16600 17500 17500 18400 18400 Deconvoluted ESI/MS of Sperm Whale Myoglobin 17332.3 100 Sp Whale Myo 0 + 1 DMPO 17443.1 100 Sp Whale Myo + DMPO + H2O2 17332.0 0 Deterding, 2005

  35. 103Y-L-E-F-I-S-E-A-I-I-H-V-L-H-S-R118 y15 y14 y13 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 LC/MS/MS of T17 of Sperm Whale Myoglobin b3 b4 b5 b8 b9 b11 DMPO (M+H) X2 100 y 6 y 7 b y y Tyr-DMPO 8 4 5 y y y 8 15 y y b y 2 11 y 3 4 y b 14 b y b 9 10 3 b 5 13 11 9 0 200 800 1400 2000 Mass Deterding, 2005

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