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.
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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 • Applications of MS to Protein Radicals • Horse Myoglobin • Sperm Myoglobin • Hemoglobin • Limitations of MS… and how to alleviate them • Summary Deterding, 2005 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 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 Electrospray Ionization Process ++ + + + Sample 3-5 ul/min 200 nl/min + + + + ESI Needle + + + + + ++ + + + Droplet Formation To MS Desolvated Ions Sampling Region Deterding, 2005 ESI Mass Spectrum of Horse Cytochrome C +16 +17 773.5 728.0 +15 825.0 +18 687.6 +14 883.9 100 Raw MS Data +13 951.7 +19 651.5 +12 1031.0 +11 +10 1124.6 1237.0 0 600 800 1000 m/z 1200 12360.5 Deconvoluted Data 100 0 10000 11000 12000 1600 1400 13000 14000 Mass Deterding, 2005 MALDI Ionization Process Sample Target Laser + + + + + + + + c + + + c + c + + + + + + + + + + + + + + + + + + Detector + + + Flight Tube Gas Phase Ions Deterding, 2005 MALDI Mass Spectrum of Horse Cytochrome C +2 6183.1 +1 12360.3 +3 4120.4 +4 2452.6 2000 5600 9200 Mass (m/z) 12800 16400 20000 Deterding, 2005 Comparison of Mass Resolution Capabilities for Intact Protein 100 12360.5 ESI/MS 0 12200 100 0 12200 12350 12360.3 12340 12500 MALDI/MS 12480 Deterding, 2005 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-1 MS-2 parent ion selection fragment ion detection Analyte Mixture Deterding, 2005 MS/MS of m/z 587.3+3 from Mixture 742.5 100 692.4 880.5 MS Scan 1043.9 587.3 1112.6 330.7 0 587.3 100 Ion Selection For MS/MS 0 100 136.1 269.1 MS/MS Scan 583.3 639.9 696.4 745.9 569.3 464.6 784.4 0 200 400 600 800 1000 1200 1400 1600 1800 m/z Deterding, 2005 MS/MS of m/z 587.3+3 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 b11 100 b10 b12 b9 ITyr y2 b6 b2 b8 b5 y3 (M+H)+ b13 b7 y8 b4 0 200 400 600 800 1000 1200 1400 1600 Deterding, 2005 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 Formation of Protein Radical Adducts Deterding, 2005 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 Only Data Dependent Acquisition MS and MS/MS info LC/MS/MS of Targeted Masses Deterding, 2005 DMPO-Spin Trapping on Horse Myoglobin ESR Analysis 50 mM Mb Deterding, 2005 X-Ray Structure of Horse Myoglobin Deterding, 2005 Deconvoluted ESI/MS of Horse Myoglobin 16952.0 HH Myo 100 0 16500 100 0 16500 17000 16951.6 +1 DMPO 17061.6 17000 17500 HH Myo + DMPO + H2O2 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 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 81.07 177.43 112.92 80.00 79.66 213.19 0 Time 50.00 100.00 150.00 200.00 Deterding, 2005 LC/ESI/MS Analysis of Tryptic Digest of Radical Adducts 941.3 100 112.9 min 942.3 650.2 % 368.2 227.1 471.2 311.1 637.1 651.2 735.4 943.3 1273.3 0 100 803.8 536.2 153.5 min 804.8 % 537.2 322.2 416.1 805.3 1607.6 666.8 0 975.3 100 227.1 372.0 % 796.8 464.4 344.0 464.7 470.2 927.3 618.5 797.7 619.2 893.8 660.6 748.3 211.1 167.9 min 976.3 (T16+DMPO+2H)2+ 976.8 1033.4 1592.6 1255.8 0 m/z 200 400 600 800 1000 1200 1400 1600 1800 Deterding, 2005 LC/MS/MS of T16 of Horse Myoglobin b3 103 b5 b7 b9 b10 b11 b12 b13 b14 b15 Y-L-E-F-I-S-D-A-I-I-H-V-L-H-S-K118 DMPO y14 y13 y11 y10 y9 y7 y6 y5 y4 y3 + (M+H) 2+ 100 (M+2H) X20 -DMPO Tyr-DMPO y3 y4 0 200 b3 y5 y6 b 5 y7 b7 y9 b9 y11 b10 y10 800 y13 y14 b b11 12 b13 b14 b15 1400 2000 Mass 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 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 kDa B C D 188 188 98 98 62 catalase trimer dimer 62 49 tetramer trimer 38 dimer 49 38 28 28 17 monomer 17 monomer 14 14 6 6 3 3 Silver Stain kDa Western Analysis Deterding, 2005 ESI/MS of Hemoglobin Samples Alpha Chain 15128.5 Hb Control Beta Chain 15869.0 100 0 mass 14600 14800 15000 15200 15400 15600 15800 16000 16200 16400 Hb + DMPO + H2O2 + 1 DMPO 15239.8 + 1 DMPO 15980.6 100 15869.1 15128.8 0 mass 14600 14800 15000 15200 15400 15600 15800 16000 16200 16400 Deterding, 2005 X-Ray Structure of Hemoglobin Tetramer Cys93 His20 Alpha chain – cyan Beta chain – purple Heme - red Tyr42 Tyr24 Tyr24 Tyr42 His20 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 LC/MS/MS of T4 of Alpha Chain of Hemoglobin b2 17 100 x2 b4 b5 V-G-A-H-A-G-E-Y-G-A-E-A-L-E-R31 y14 y13 y12 y11 y10 y9 y8 y7 DMPO y1 0 100 * y* * 4 b4 200 300 500 b2 17 100 x2 * y5 400 600 700 b4 b5 800 900 Mass y12 * y12 –DMPO -H2O y8 1000 1100 1200 1300 1400 1500 V-G-A-H-A-G-E-Y-G-A-E-A-L-E-R31 DMPO b4 b5 y4 His-DMPO y1 200 * 300 400 500 y7 y8 800 900 Mass y12 y11 y12 –DMPO -H2O y5 700 (M+H)+ y6 y5 y4 y3 y2 y1 -DMPO -H2O y10 (M+2H)2+ 600 1600 b6 b7 b8 y14 y13 y12 y11 y10 y9 y8 y7 0 100 y6 y5 y4 y3 y2 y1 -DMPO -H2O (M+2H)2+ y7 (M+H)+ 1000 1100 1200 1300 1400 1500 1600 Deterding, 2005 X-Ray Structure of Hemoglobin Tetramer Cys93 His20 Alpha chain – cyan Beta chain – purple Heme - red Tyr42 Tyr24 Tyr24 Tyr42 His20 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 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 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 Complementary Detection of Protein Radicals Mass Spec Anti-DMPO Ab ESR Protein Id Yes No No Amino Acid Id Yes No Yes Specific residue Yes No No Sensitivity ug ug mg Selectivity No Yes Yes Deterding, 2005 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 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 LC/MS/MS of T10-11 of Beta Chain of Hemoglobin b2 b3 b4 b5 83 b10 b17 G-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 y1 (M+H)+ DMPO y5 100 y3 -DMPO -H2S y16-DMPO-H2S y8 y1 y6 b3 b5 y16 y9 y10 y11 y7 y14 y12 y13 y18 y17 y20 y19 0 200 400 600 800 1000 1200 1400 Mass 1600 1800 2000 2200 2400 2600 Deterding, 2005 LC/MS/MS of T6 of Alpha Chain of Hemoglobin b2 b3 41 100 x2 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 T-Y-F-P-H-F-D-L-S-H-G-S-A-Q-V-K56 y15 y14 y13 y12 y11 y10 y9 y8 y7 y6 (M+H)+ y5 y4 y3 y2 y1 DMPO b7 y6 b14 -H20 y7 b 5 y8 y1 y2 b2 y9 b3 y10 y11 y12 y13 y14 b12 y15 0 200 400 600 800 1000 1200 1400 1600 1800 Mass Deterding, 2005 Deconvoluted ESI/MS of Sperm Whale Myoglobin 17332.3 100 Sp Whale Myo 0 16600 17500 18400 + 1 DMPO 17443.1 100 Sp Whale Myo + DMPO + H2O2 17332.0 0 16600 17500 18400 Deterding, 2005 LC/MS/MS of T17 of Sperm Whale Myoglobin b3 b4 b5 b8 b9 b11 103 118 Y-L-E-F-I-S-E-A-I-I-H-V-L-H-S-R DMPO 100 y15 y14 y13 y11 y10 y9 y8 y7 y6 y5 y4 y3 y2 (M+H) X2 y6 y7 Tyr-DMPO y2 y 3 0 200 y4 y5 b3 b4 y8 b5 800 b8 y9 y10 Mass y11 b9 b11 y13 1400 y14 y15 2000 Deterding, 2005