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ROXY™ EC/MS System

ROXY™ EC/MS System. Service Training 2012. Introduction to ROXY EC system basics & application review ReactorCell and µ-PrepCell maintenance filling the µ-PrepCell Dialogue training program concept practical workshop including event programming ROXY EC system Installation

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ROXY™ EC/MS System

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  1. ROXY™ EC/MS System

  2. Service Training 2012 • Introduction to ROXY EC system • basics & application review • ReactorCell and µ-PrepCell maintenance • filling the µ-PrepCell • Dialogue training • program concept • practical workshop including event programming • ROXY EC system Installation • establishing communication with RS232 cable • trigger cable • grounding kit • Practical insights on application • test compounds • mass spectrometric analysis • optimization of conditions

  3. Application Areas Electrochemistry/MS

  4. Electrochemistry up front MS Instrumental set-up ROXY EC System ReactorCell™ µ-PrepCell™

  5. Electrochemistry (EC) upfront MS Instrumental set-up ROXY™ EC System ROXY™ EC/LC System ROXY™ EC/LC System

  6. Electrochemistry upfront MS Drug/xenobiotic metabolism

  7. Electrochemistry upfront MS Prediction of drug/xenobiotic metabolism ReactorCell (or µ-PrepCell) 3-D MS Voltammogram Drug 2-D MS Voltammogram 7

  8. Electrochemistry upfront MS Prediction of drug/xenobiotic metabolism Amodiaquin metabolic pathway: Faber et al., Angew. Chem. Int. Ed. Engl. 5 (2011) A52-58

  9. Electrochemistry upfront MS Protein chemistry

  10. Electrochemistry upfront MS Protein chemistry Amino acid Functional group Oxidized forms, with mass change Tyrosine phenol quinol, +16 Daquinone, +14Da Tryptophan indolol, +16 Daindolone, +14Da indole Cysteine thiol sulfenic acid, +16 Dasulfinic acid, +32Da sulfonic acid, +48 Da Methionine methylsulfoxide, + 16 Da methylsulfone, + 32 Da methylthioether

  11. Drug – protein adduct formation

  12. Electrochemistry upfront MS Protein chemistry

  13. Mechanism of cleavage after Tyrosine (Tyr; Y) & Tryptophan (Trp; W) residues Tyrosine containing peptides: 1000mV Tryptophan containing peptides: 800mV • Oxidation and cleavage pathways are pH dependent: • oxidation yield decreases with increasing pH • cleavage products formed only in acidic and neutral conditions J. Roeser et al., Anal. Chem., 2010, 82 (18), 7556

  14. Cleavage of Angiotensin I (DRVYIHPFHL) • ADVANTAGES: • 1) …alternative to enzymatic digestion by electro-chemical push button reaction in seconds! • 2) clean, no enzymes, no non-specific cleavage, no auto-digestion, etc. • CURRENT STATUS: • cleavage of big proteins is under development, • optimization to increase the reaction yield.

  15. Electrochemical Disulfide Bond Reduction

  16. Tested compounds

  17. 1147.75 NL: 5.26E5 100 111214 Insulin MD pulse 01#7887-8260 RT: 90 23.22-24.32 AV: 374 T: 80 ITMS + c ESI Full ms [360.00-2000.00] 70 956.75 60 Relative Abundance 50 40 30 1434.32 850.98 20 681.12 960.56 1152.22 10 1169.72 1439.61 626.38 1213.92 1911.39 1480.74 0 NL: 3.15E5 851.07 681.18 100 insulin 90 01#3203-3903 RT: 9.37-11.41 AV: 701 T: 80 ITMS + c ESI Full ms [360.00-2000.00] 70 60 50 40 30 858.86 20 680.36 866.11 571.43 1134.04 10 1170.66 1369.11 1643.48 1812.84 0 500 1000 1500 2000 m/z Comparison with MD Insulin Insulin reduced on MD electrode Reduced Insulin Insulin reduced on new electrode No Insulin present

  18. 1147.5278 NL: 1.15E5 100 90 02#1069-1084 RT: 10.76-11.86 AV: 16 F: 80 FTMS + c ESI Full ms 70 [300.00-2000.00] 60 Relative Abundance 956.4407 50 40 30 20 960.2694 10 1160.3113 680.7421 850.9267 970.7545 1133.5199 646.0303 944.4320 751.2788 1255.4004 1361.7371 0 680.7420 NL: 6.77E4 100 90 02#1017-1029 RT: 850.6758 6.93-7.81 AV: 13 F: FTMS 80 + c ESI Full ms 70 [300.00-2000.00] 60 50 40 30 20 780.3308 1147.7295 10 956.7773 858.1774 666.9364 1211.2011 1080.6786 537.0156 917.9375 964.4340 1316.5495 0 600 700 800 900 1000 1100 1200 1300 m/z Insulin - new working electrode Cell OFF No reduction Pulse ON Complete reduction

  19. 546.5790 NL: 5.19E4 100 111216 Somatostati 90 pulse 02#152-163 RT: 80 4.78-5.29 AV: 12 F: FTMS + c ESI Full ms 70 559.2278 [100.00-2000.00] 60 819.3646 Relative Abundance 50 40 517.1583 30 571.8769 20 838.3381 419.6727 296.9704 10 385.9019 474.8328 615.5303 706.8891 749.4350 1008.4596 1140.0590 1241.1783 NL: 2.43E4 0 547.2509 100 111216 Somatostati 90 pulse 02#96-112 RT: 80 2.14-2.89 AV: 17 F: FTMS 820.3723 + c ESI Full ms 70 [100.00-2000.00] 60 50 40 30 559.8999 279.9241 20 510.2107 439.8490 10 835.3762 706.8571 567.5920 784.0946 320.9508 926.6889 986.0088 1129.5707 1236.7040 0 300 400 500 600 700 800 900 1000 1100 1200 m/z SomatostatinNew working electrode Z = 3 Cell OFF No reduction Z = 2 Z = 3 Pulse ON Complete reduction Z = 2

  20. Cell OFF No reduction 1576.32 NL: 8.28E4 100 111216 Lactalbumin 90 pulse01#772-811 RT: 80 9.41-11.21 AV: 40 F: 70 1575.98 FTMS + c ESI Full ms 1773.10 60 Relative Abundance [800.00-2000.00] 50 1772.98 40 1580.53 1418.69 1777.97 30 1782.84 20 1289.90 864.71 1792.08 1182.57 10 994.67 1593.07 1420.38 1807.05 1290.44 878.72 998.66 1615.27 1910.60 0 1183.16 NL: 1.11E4 100 1092.22 Pulse ON Complete reduction 111216 Lactalbumin 90 pulse01#887-921 RT: 1182.91 80 14.69-16.24 AV: 35 F: 1092.30 70 FTMS + c ESI Full ms 1184.41 1014.28 60 946.80 [800.00-2000.00] 1290.63 50 1412.87 1290.36 40 1576.99 1094.53 1186.41 887.68 30 1017.06 1577.21 1419.39 949.12 1293.35 20 1422.29 1581.42 1935.86 10 1181.74 1526.19 1783.73 1233.91 1839.65 1302.63 1614.94 1959.88 0 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z α – LactalbuminNew working electrode Electrochemical reduction of the protein results in shift of charge state distribution suggesting conformational change of protein (S-S bridges reduction).

  21. Electrochemical disulfide bond reduction • on-line, electrochemical disulfide bond reduction with DESI MS • identification of disulfide containing peptides from enzymatic digestion mixture • derivatization of thiols by selenamid • charge state distribution in proteins • (native vs. reduced) Zhang et al., J. Proteome Res., 2011, 10, 1293

  22. Electrochemical Desalting of Proteins Poster at BSPR, Cambridge Mohamed Benama 0 V 2.8 V Deconvoluted MS at 0V and 2.8V showing protein desalting. correspond to [Na+ + K+] combination correspond to background formylation of the protein

  23. Electrochemical Oxidation as a Surface Mapping Probe of Higher Order Protein Structure Cell OFF Cell ON 23 McClintock et al., Anal. Chem. 2008, 80, 3304

  24. Electrochemistry upfront MS Oxidative damage of DNA

  25. Oxidative Damage of DNA 25

  26. Laborious, time-consuming and hardly automatable Stability of the (modified) nucleic acids during sample prep Low specificity and sensitivity Oxidative Damage of DNA 26

  27. Oxidative Damage of DNA 27

  28. Mass Voltammograms of Nucleosides Herbert Oberacher, Institute of Legal Medicine, Innsbruck, Austria; Electrochemical Simulation of Oxidation Processes Involving NucleicAcids On-line Monitored with Electrospray Ionization-Mass Spectrometry - poster IMSC 2009 28

  29. Several studies on cell cultures and rodents have demonstrated that acetaminophen can covalently bind to nucleic acids after metabolic activation. Oxidative Damage of DNA EC/MS of guanosine +APAP: EC/MS of guanosine:

  30. Oxidative Damage of DNA Electrochemistry upfront MS provides new tool to asses the antioxidant potency of chemicals ! 30

  31. Electrochemistry upfront MS 31

  32. Signal enhancement LC–EC–UV/VIS and LC–EC–MS chromatograms for a mixture of the sixteen priority pollutant PAH. Positive-ion mode: naphthalene (1), acenaphthylene (2), acenaphthene (3), fluorene (4), phenanthrene (5), anthracene (6), fluoranthene (7), pyrene (8), benzo[a]anthracene (9), chrysene (10), benzo[b]fluoranthene (11), benzo[k]fluoranthene (12), benzo[a]pyrene (13), dibenzo[a,h]anthracene (14), benzo[ghi]perylene (15), indeno[1,2,3-cd]pyrene (16). (a) UV chromatogram recorded at 254 nm; (b) MS chromatogram, scan mode m/z 150–400, electrochemical flow cell off; (c) MS chromatogram, scan mode m/z 150–400, electrochemical flow cell 1.6 V; (d) MS chromatogram, selected ion monitoring (SIM) mode, electrochemical flow cell 1.6V Anal. Bioanal. Chem. 378 (2004) 917– 925

  33. Electrochemistry upfront MS Skin sensitization

  34. Electrochemistry upfront MS

  35. Electrochemistry upfront MS Environmental analysis

  36. Persistence in environment, stability in EC? => QSAR modeling…

  37. Stability and structure Compound StructureEC starting voltage (mV) Naphthalene 340 Anthracene 930 Phenanthren 600 Benzo[a]anthracene 750 Tetracene 1600 Chrysene 1300

  38. Electrochemistry upfront MS 38

  39. Electrochemistry upfront MS Other applications Joint Conference of Germanand Polish Mass Spectrometry Society Poznan, Poland March 4 - 7, 2012

  40. Electrochemistry upfront MS Other applications This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LCHR-MS.

  41. Summary EC/MS represents a powerful technique for study of REDOX reactions in life science

  42. Thank you!

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