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Session Overview

MS/MS Spectral Interpretation Linda Breci Chemistry Mass Spectrometry Facility University of Arizona MS Summer Workshop. MS/MS Spectral Interpretation small molecule structure Arpad Somogyi Chemistry Mass Spectrometry Facility University of Arizona MS Summer Workshop. Session Overview.

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Session Overview

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  1. MS/MS Spectral InterpretationLinda BreciChemistry Mass Spectrometry FacilityUniversity of Arizona MS Summer Workshop

  2. MS/MS Spectral Interpretationsmall molecule structureArpad SomogyiChemistry Mass Spectrometry FacilityUniversity of Arizona MS Summer Workshop

  3. Session Overview • Ways to approach predicting fragment ion formation • Fragmentation examples • Peptides • Fragmentation mechanism • Sequence a peptide • Flavonoids • Fatty Acids • Oligonucleotides

  4. MS/MS Fragmentation Few libraries, little software available for data analysis • Why?

  5. We need useful information from MS/MS spectra Few libraries, little software available for data analysis • Why? For MS/MS you have at least one of each of these: • Ionize • EI • CI • ESI • NSI • MALDI • FAB • Activate • CID • SID • SORI • IRMPD • ECD • BIRD • Analyze • Q • Q-trap • linear-trap • B sectors • E sectors • FTICR • TOF

  6. You put them together like this: * ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF * NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF * EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q * MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESI-EBSector-CID-EBSector * and on…and on…

  7. You put them together like this: * ESI-CID-Q-trap * ESI-SORI-FTICR * FAB-EBSector-SID-TOF * NSI-CID-Q-trap * MALDI-TOF-CID-TOF * NSI-Linear-trap-CID-FTICR * NSI-Q-trap-SID-TOF * EI-CID-Q-trap * ESI-IRMPD-FTICR * ESI-Q-CID-Q * MALDI-TOF-CID-TOF * NSI-BIRD-FTICR * ESI-EBSector-CID-EBSector * and on…and on… • Different source designs • Example: ESI capillary temperature • Different analyzer designs • Example: Gas pressure, length of ion path (D timeframe) And you buy them from different manufacturers

  8. How ions will fragment must be considered from fundamentals (rather than rules) • Ways to approach predicting MS/MS fragment formation • Literature • Study methods and ID’d spectra for your ion class • Likely sites of protonation (or deprotonation) • Find proton affinities or acid strengths • Mobility of protons • Consider the likelihood of multiple cleavage sites • Consider multiple gas-phase configurations • Likely leaving groups

  9. Today’s Topic Types of ions formed • EI (hard ionization) • M+·Radical ion • A lot of fragmentation occurs upon ionization • CI, FAB, ESI, APCI, MALDI (soft ionization) • [M+H]+ Protonated ion • [M-H]- Deprotonated ion • [M+Na]+ and other metal cations

  10. EI is not an MS/MS method • Discussed Day 4 • Libraries of EI spectra are useful • NIST/EPA/NIH Mass Spectral Library with Search http://webbook.nist.gov/chemistry/ • Libraries are not always helpful, tutorials available • http://www.chem.arizona.edu/massspec/

  11. Today’s Topic 2 Categories of fragments from protonated or deprotonated molecules (CI, FAB, ESI, APCI, MALDI) • Charge Remote • Fragmentation reactions uninfluenced by charge • High energy process • Charge remote references provided • Charge Directed • Bond cleavage occurs with involvement of charge • Low energy • Most informative for many molecules

  12. How ions will fragment must be considered from fundamentals (rather than rules) • Literature • Study methods and ID’d spectra for your ion class • Likely sites of protonation (or deprotonation) • Find proton affinities or acid strengths • Mobility of protons • Consider the likelihood of multiple cleavage sites • Consider multiple gas-phase configurations • Likely leaving groups

  13. How ions will fragment must be considered from fundamentals (rather than rules) • Literature • Study methods and ID’d spectra for your ion class • Likely sites of protonation (or deprotonation) • Find proton affinities or acid strengths • Mobility of protons • Consider the likelihood of multiple cleavage sites • Consider multiple gas-phase configurations • Likely leaving groups

  14. Fragmentation is a multi-step process Step #1: Create Ions (add 1 or more protons) ELECTROSPRAY

  15. Fragmentation is a multi-step process Step #1: Create Ions (add 1 or more protons) ELECTROSPRAY Step #2: Add energy (activation) SID CID

  16. Fragmentation is a multi-step process Step #3: Charge Directed Cleavage Neutral + Fragment ion What are the likely sites of proton location?

  17. Model possible sites of proton location(or loss of H) in Serine • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M)

  18. Model possible sites of proton location(or loss of H) in Serine Model with CH3NH2 (methyl amine) • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M)

  19. PA DH acid methyl amine 214.9 402.0 Model possible sites of proton location(or loss of H) in Serine Model with CH3NH2 (methyl amine) • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M) Ref: NIST

  20. PA DH acid methyl amine 214.9 402.0 acetic acid 187.3 348.1 Model possible sites of proton location(or loss of H) in Serine Model with CH3COOH (acetic acid) Model with CH3NH2 (methyl amine) • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M) Ref: NIST

  21. PA DH acid methyl amine 214.9 402.0 acetic acid 187.3 348.1 methanol 180.3 382.0 Model possible sites of proton location(or loss of H) in Serine Model with CH3COOH (acetic acid) Model with CH3NH2 (methyl amine) Model with CH3OH (methanol) • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M) Ref: NIST

  22. PA DH acid methyl amine 214.9 402.0 acetic acid 187.3 348.1 methanol 180.3 382.0 Model possible sites of proton location(or loss of H) in Serine Model with CH3COOH (acetic acid) Model with CH3NH2 (methyl amine) Model with CH3OH (methanol) Sites of Likely protonation: NH2 > COOH > OH deprotonation: COOH > OH > NH2 • M + H → [M+H]+DHrxn = -PA (M) • M → [M - H]- + H+DHrxn = DHacid (M) Ref: NIST

  23. How ions will fragment must be considered from fundamentals (rather than rules) • Literature • Study methods and ID’d spectra for your ion class • Likely sites of protonation (or deprotonation) • Find proton affinities or acid strengths • Mobility of protons • Consider the likelihood of multiple cleavage sites • Consider multiple gas-phase configurations • Likely leaving groups

  24. Proton mobility • Intramolecular proton transfer influences • number of site-directed fragmentations • amount of energy required for fragmentation • Intramolecular proton transfer affected by • site basicity • gas-phase configuration • Examples that follow: • Spectra of increasingly basic peptides • Overview chart demonstrating proton mobility (or lack of) • Spectra of peptide conformers

  25. Compare Gas Phase Basicity Arg (R): 240.6 kcal/mol Lys (K): 227.3 kcal/mol His (H): 227.3 kcal/mol 50 eV (SID) 40 eV (SID) 40 eV (SID) Ref: Gu, 1999

  26. Pairwise bond cleavage between amino acids (Xxx-Zzz)

  27. Peptides with more basic Arg (R) vs. Lys (K) .............R 1+ .............K

  28. Prediction based on model peptides: Selective Cleavage at Asp-Xxx will depend on number of “Mobile” Protons Huang, Wysocki, Tabb, Yates Int. J. Mass Spectrom. 219, (1), 233-244, 2002

  29. Peptides with basic Arg (R) 1 proton vs. 2 protons 1+ .............R 2+

  30. Gas-phase conformation influences MS-MS spectra observed Ala-Ala-Pro-Ala-Ala Most Natural occurring amino acids have L configuration at the chiral center (stereospecific biosynthesis)

  31. Calculated structure of [AAPAA + H]+Many sites of possible interaction No solvent in the gas phase!

  32. Gas-phase confirmation can influence MS-MS spectra observedPeptides containing proline stereoisomers fragment differently All L-amino acids except central residue AVDPLG All L-amino acids

  33. Gas-phase confirmation can influence MS-MS spectra observedPeptides containing proline stereoisomers fragment differently All L-amino acids except central residue AVDPLG All L-amino acids

  34. Gas-phase confirmation can influence MS-MS spectra observedPeptides containing proline stereoisomers fragment differently All L-amino acids except central residue AVDPLG All L-amino acids

  35. Statistical analysis of cleavage at the Xxx-Pro bond Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971

  36. Statistical analysis of cleavage at the Xxx-Pro bond Asp, His = Selective cleavage residues Val, Ile, Leu = Bulky aliphatic side chains Breci, Tabb, Yates, Wysocki, (2003) Analytical Chem. 75:1963-1971

  37. How ions will fragment must be considered from fundamentals (rather than rules) • Literature • Study methods and ID’d spectra for your ion class • Likely sites of protonation (or deprotonation) • Find proton affinities or acid strengths • Mobility of protons • Consider the likelihood of multiple cleavage sites • Consider multiple gas-phase configurations • Likely leaving groups

  38. Likely Leaving Groups • Bond cleavage is dependent on various factors including: • Leaving Groups • Neighboring group participation reactions • Intermediates (ion-neutral complex) • For [M+H]+ ions the leaving group is a neutral • lower methyl cation affinity is one measure of likelihood • Compilations available in the literature • Related to proton affinity

  39. (kcal/mol) Ref: Bartmess, 1989

  40. Proton Affinity vs. Methyl Cation Affinity Ref: Bartmess, 1989

  41. Some fragmentation studies & basics • Few examples from literature • Cannot talk about all classes of compounds • These examples suggest problem solving approaches • Examples: • Peptides • Fragmentation mechanism • Sequence a peptide • Flavonoids • Fatty Acids • Oligonucleotides

  42. Peptides • Product ion spectra contain many types of fragment ions • charge directed • charge remote • internal fragments • immonium ions • Important for sequencing • amino acid determined from D mass between peaks in spectrum • “y” ions series • “b” ions series • immonium ions (identify amino acids in the peptide) • “a” ions (confirm “b” ion after a loss of CO, 28 amu) • Presented here: • peptide fragment ions • a mechanism for fragment ion formation • a peptide to sequence

  43. Peptide fragment ions c2 Peptide bond fragment ions b2 a2 z2 y2 x2 Internal immonium ion Amino acid immonium ion

  44. Protonation occurs at amide oxygen or nitrogen Ref: Yalcin, 1996

  45. Protonation occurs at amide oxygen or nitrogen Ref: Wysocki, 2000

  46. A mechanism of peptide fragmentation (1) D positive charge (2) Nucleophilic attack Ref: Wysocki, 2000

  47. A mechanism of peptide fragmentation (1) D positive charge (2) Nucleophilic attack (3) cyclic intermediate Ref: Wysocki, 2000

  48. A logical mechanism of peptide fragmentation (3) cyclic intermediate (4) Rearrangement Ref: Wysocki, 2000

  49. A logical mechanism of peptide fragmentation + b oxazolone ion neutral Ref: Wysocki, 2000

  50. A logical mechanism of peptide fragmentation + oxazolone neutral (or other structure) y ion Ref: Wysocki, 2000

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