1. CH 908: Mass Spectrometry Lecture 9 Electron Capture Dissociation of Peptides and Proteins Prof. Peter B. O’Connor

2. Objectives for this lecture Odd vs. Even electron fragmentation reactions First paper History of the mechanisms –hot hydrogen model –UW model –Odd results noticed with polymers and zinc-binding peptides –Radical cascade mechanism cyclic peptide data radical migration double resonance radical traps Utility –Disulphide bonds –Protein structural studies –Labile modifications –Isomer determination –HDX? Other ExD methods

3. Odd vs. Even Electron Fragmentation Even electron = proton rearrangements Odd electron = radical rearrangements Non-ergodic fragmentation = FAST!!

4. ECD Spectrum

6. Hot H Mechanism of ECD

7. UW Mechanism

8. Activated-Ion ECD

11. Kjeldsen, F.; Haselmann, K. F.; Budnik, B. A.; Jensen, F.; Zubarev, R. A. Dissociative capture of hot (3-13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation Chem Phys. Lett. 2002, 356, 201-206. “Hot” ECD

14. The effect of metals In this case, Zinc binding killed the electron – thus killing fragmentation

15. Electron capture dissociation of polyethylene glycol HO-(CH2-CH2-O) n -H

16. Cyclic peptide structures cyclo-LLFHWAVGHgramicidin Scyclosporin A Two histidines Tryptophan phenylalanine Ornithine Proline Phenylalanine N-Methylated Two unusual Amino acids Leymarie, N.; Costello, C. E.; O'Connor, P. B. Electron capture dissociation initiates a free radical reaction cascade J Am Chem Soc 2003, 125, 8949-8958.

17. ECD of cyclo- LLFHWAVGH (M+2H) 2+ 3004005006007008009001000 m/z X30        X12 cleavage assignable to backbone cleavage cleavage assignable to loss of H, H 2 O, NH 3, CO, CONH cleavage assignable to sidechain fragmentation † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † † †  : electronic noise,  Harmonic

18. The free radical reaction cascade will continue until one of the following occurs: 1.The various energy losses in the reactions and in black body radiation cool the system sufficiently that further rearrangments are impossible 2.The free radical is eliminated leaving the charge containing peptide in an even-electron state 3.The free radical is stabilized at a site of low reactivity

19. Is the Free Radical Cascade mechanism really correct? 1.FRC mechanism implies radical migration. Calculations suggest radical will migrate to the alpha carbon. If so, deuterium labeling those (non exchangeable) positions should cause the fragments to show D scrambling 2.FRC mechanism implies that the radical intermediates are long lived (≈ microseconds or more). This can be tested by double resonance. 3.FRC mechanism implies radical migration. Addition of a radical trap moiety to the peptide should radically change the fragmentation.

20. Free Radical Cascade in Linear Peptide ECD: Deuterium scrambling by Hydrogen Abstraction C R 1 CH C NH CH R 2 C NH D C D O O O R 1 CD C NH CH R 2 C NH CD C O O O H R 1 CD C NH CH R 2 C NH CD C H O O O CD C H O R 1 CD C NH CH R 2 C NH O O +  mass = -1.006 Da CD C D O secondary primary Deuterium substitution at sites where the radical migrates to Isotopic scrambling prior to secondary cleavage

21. ECD of D-labeled synthetic peptides: H/D Scrambling 10551060 10651070 10651070 855860 865 860865 630635 630 635 625630 1240 1245 1250 1255 12501255 515520 515 520 510515 805810 805810 800805 985 990 9951000 995 1000 690 695 690 695 685690 1170 1175 11801185 11801185 935 940 935 940 925930935 440445 440 445 440445 c 15 c 14 c 13 c 12 c 11 c 10 c9c9 c8c8 c7c7 c6c6 c5c5 Top: ECD of BUSM 1 Middle: 0.2 eV ECD of BUSM 2 Bottom: 9 eV ECD of BUSM 2 Spectra of BUSM 2 were shifted left by 2 Daltons per glycine to align with those of BUSM 1 RAG 2D ADG 2D DADG 2D DAG 2D AAR BUSM 1 = undeuterated BUSM 2 = deuterated

22. H/D Scrambling Results The initial radical does migrate to α- carbons The best mechanism supports a hydrogen bonded dimer as the long-lived radical intermediate

23. Is the Free Radical Cascade mechanism really correct? 1.FRC mechanism implies radical migration. Calculations suggest radical will migrate to the alpha carbon. If so, deuterium labeling those (non exchangeable) positions should cause the fragments to show D scrambling 2.FRC mechanism implies that the radical intermediates are long lived (≈ microseconds or more). This can be tested by double resonance. 3.FRC mechanism implies radical migration. Addition of a radical trap moiety to the peptide should radically change the fragmentation.

24. Double Resonance + + + + + Resonantly Eject Timeframe = 0.01 – 1 msec X X = long lived = short lived

25. Double Resonance inject ions quenchisolateECDExciteDetect Resonant Excite Fragments derived from long-lived intermediates will disappear from spectra Fragments from short-lived intermediates will not change Which fragments come from long lived intermediates?

26. 30060090012001400400500700800100011001300 22 c4c4 * c5c5 M 2+ c6c6 a7a7 c7c7 c8c8 z9z9 c9c9 c 10 [M+2H] + Substance P ECD RPKPQQFFGLM-NH 2 30060090012001400400500700800100011001300 *: electronic noise m/z

27. Substance P z9z9 c4c4 NH NH 2 HN NH 2 O H N O N N O NH 3 + O N H H ONH 2 O H N NH 2 O O N H O H N O NH OHN O N H O NH 2 S

28. 300400500600700 c3c3 22 a4a4 c4c4 a5a5 c5c5 a6a6 c6c6 z6z6 a7a7 z7z7 b7b7 y7y7 c7c7 a7a7 c8c8 BUSM 1 ECD RAAA GADG DGAG ADAR 80090010001100120013001400 z8z8 a9a9 z9z9 b9b9 y9y9 c9c9 a 10 c 10 a 11 c 11 a 12 c 12 a 13 z 12 c 13 z 13 a 14 b 14 z 14 c 14 a 15 z 15 c 15 -NH 3 [M+2H] + -CO -34 -45 -60 -72 -78 -104/5 -101 -130 -149 m/z

29. Is the Free Radical Cascade mechanism really correct? 1.FRC mechanism implies radical migration. Calculations suggest radical will migrate to the alpha carbon. If so, deuterium labeling those (non exchangeable) positions should cause the fragments to show D scrambling 2.FRC mechanism implies that the radical intermediates are long lived (≈ microseconds or more). This can be tested by double resonance. 3.FRC mechanism implies radical migration. Addition of a radical trap moiety to the peptide should radically change the fragmentation.

30. Radical traps

32. Fixed Charge Derivatives

34. Ok, so what good is it?

35. ECD and Disulfide bonds

37. Native ECD

38. Unfolding probed by ECD yield

44. Other labile modifications mapped by ECD Posphorylation N-glycosylation O-glycosylation Sialic acid residues on oligosaccharides Sulfation Hydrogen bonds – noncovalent interactions

45. asparagine aspartic acid isoaspartic acid or β-aspartic acid The cannonical mechanism of deamidation involves cyclization of asparagine (Asn) to form the succinimide intermediate with loss of ammonium, followed by hydration at either amide bond to form a mixture of aspartic (Asp) and isoaspartic (isoAsp) acids. Deamidation is irreversible under physiological conditions, but the isomerization of the products occurs at a relatively slow rate. Asparagine Deamidation

46. Aspartic Acid Mass/Charge (m/z) C 6. +58 m/z = 572.3031 572.0574.0576.0743.0745.0747.0 1115.01117.01119.0 572.0574.0576.0 743.0745.0747.0 1115.01117.01119.0 C 8. +58 m/z = 744.3515 C 13. +58 m/z =1115.4956 # # Isoaspartic Acid c n +58z l-n -57 isoAspartyl c n +58 fragment ion # indicates secondary fragment ion due to loss of NH3 and CHON from y14 (found in the spectra of both peptides) RAAAGADGDGAGADAR ABCABC ZYZY

47. Deamidated Tryptic Peptide of Cytochrome C m/z 585586587 Before incubation 3 Days 7 Days 12 Days 28 TGPNLHGLFGR 38, 2 +  D/isoD Asn 31 of Cytochrome C is not exposed to the solvent and cannot deamidate in the proteins native state. The tryptic peptide 28 TGPNLHGLFGR 38 was found in the digest of Cyt. C. The digest was incubated for 2 weeks at 37°C and pH 11 (20mM CAPS titrated w/ NH 4 OH) to fully deamidate Asn 31. The peptide (cleaned w/ POROS) was then isolated and subjected to ECD. cytochrome C Asn Asp

48. ~ 5007501000125015001750 m/z w3w3 c3c3 z 3 y3y3 z 4 z 5 - (leucine side chain) c4c4 z 5 y5y5 c5c5 z 6 y6y6 c6c6 z 14 2+ y7y7 c7c7 c8c8 (M+2H) 2+ z 8 y8y8 z 9 y9y9 c9c9 z 10 c 10 z 11 y 11 c 11 z 12 c 12 c 13 z 13 c 14 c 15 (M+3H) 3+ x7 1021.532 (~1 ppm) 1020.51022.51024.5 m/z 736.337 (~1 ppm) 736737738 m/z c 6+58 z 10 -57 ECD of deamidated Calmodulin tryptic peptide V F D K D G D G Y I S A A E L RV F D K D G D G Y I S A A E L R a 10 z y c a z 10 -57 c 6 +58 60 Da loss  D 3, D 5 848.5849.5850.5 m/z (M+3H)2 + - Asp side chain 848.926 (~1 ppm) † loss of NH 3 and CO 2 from (M+3H)2 +. Note: internally calibrated on [M+3H] 3+, [M+2H] 2+, z 12 and their isotopes. † c 4+58 D95 isoD95? z 12 -57 D95 isoD95? z 14 -57 D93 isoD93?

49. Abundance ratio of Asp/isoAsp diagnostic peaks

50. Abundance ratios are linear Plots of the z n -57 relative abundance versus % isoAsp content for; ▲, YWQHTADQFR-NH2 ; ♦, WAFDSAVAWR-NH2 ; ■, YDFIEYVR-NH2. One calibration point is always available.

51. “ECD” on an ion trap

52. Electron-ion reaction methods… Electron Capture Dissociation (ECD) Activated Ion ECD (AI-ECD) Hot ECD Electron Transfer Dissociation (ETD) Electron Detactment Dissociation (EDD) Electron Ionization Dissociation (EID or EIEIO) Electron Capture Induced Dissociation (ECID)

53. Self Assessment How does ECD work? What fragments do you expect (mostly) from proteins and peptides? Why is it important to track the position of the radical? How can ECD be used to differentiate isomeric amino acids (2 examples)? Why do ECD rather than CAD?

54. Fini… CH908: Mass spectrometry Lecture 1