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Computer Assisted Sequencing of Cyclic Peptides

Computer Assisted Sequencing of Cyclic Peptides. James E. Redman Keith M. Wilcoxen M. Reza Ghadiri. Antimicrobial Cyclic Peptides. Head-to-tail D , L -cyclic peptides Cationic & amphipathic  antimicrobials Membrane active Sequence dependence Selectivity

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Computer Assisted Sequencing of Cyclic Peptides

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  1. Computer Assisted Sequencing of Cyclic Peptides James E. Redman Keith M. Wilcoxen M. Reza Ghadiri

  2. Antimicrobial Cyclic Peptides • Head-to-tail D,L-cyclic peptides • Cationic & amphipathic  antimicrobials • Membrane active • Sequence dependence • Selectivity • Microbial vs. mammalian membranes • Library synthesis & screening • Construction of QSAR

  3. Peptide Synthesis

  4. Split-and-Pool Synthesis • Single bead - Single compound • Macrobead resins • 500 mm diameter • Dispense into plates & cleave peptide • Sufficient compound for biological testing

  5. Peptide Identification • Edman degredation • Requires N-terminus • Mass spectrometry • CID & MSn • Complicated CID spectra for cyclic peptides • Multiple ring opening sites

  6. Example • c[KWLWKS] • MS/MS of pure material

  7. Requirements • MS sequencing of cyclic peptides • No chemical encoding • Complicates synthesis • Contaminates products • Automated analysis of mass spectra • Cyclic peptide fragmentations • Library residue constraints • Software integration • Visualization • Archival

  8. Amino acids Sequence list Computed MS Parent Ion MS Compare & score Sequence shortlist Computed MSn Ranked sequences Compare & score Fragment MS Software Outline

  9. Calculation of Spectra • Calculation of CID spectra • Backbone fragmentations • Cleavage of amide bond • Loss of CO • Side chain losses • NH3 from Arg, Lys, Gln • H2O from Ser, Thr, Glu • MSn • Iterative filtering

  10. Uses raw data Predicted  Experimental Sum over j predicted peaks wj = scoring weight Ij= maximum experimental intensity Normalization “Critical analysis” Pairwise scoring Use unique peaks only Reveals potential ambiguities Scoring

  11. Limitations • Isobaric sequences • Retro sequences • Enantiomeric / diastereomeric sequences • Ile / Leu • Lys / Gln • Simplified calculations • Focus on commonly observed fragmentations • Approximations for side chain loss • Multiple charging for parent ion only

  12. User Interface • Server application • Compiled C++ library • No UI • Fully scriptable • Integrates with Windows applications • Excel interface • Visual Basic for Applications • Worksheet input/output • Custom menus

  13. Excel Interface • Sequence menu • Parent spectrum parameters • CID spectrum parameters • Library composition

  14. Validation • Test compounds • Known sequences • Purified compounds • Cyclic-D,L-hexapeptides • Cyclic-D,L-octapeptides • Hypothetical split & pool library • Mass spectra • Sonic spray ionization • Ion trap MS and MS/MS

  15. Cyclic Hexapeptides • 11 Pure compounds • c[KWLWKE], c[KWLWKF], c[KRWLWL] etc. • Hypothetical library (1.8  106 sequences) • Fixed Lys residue • 5 variable residues • 18 Possibilities • Omit Q, I • Manually optimized MS/MS

  16. Cyclic Hexapeptides • Software output • Shortlist - sequences with correct parent mass • Top scoring sequence • Sequence selected by “critical analysis” • Hundreds of sequences shortlisted • Allowing for Q/K and retro sequences... • Scoring rank • 10/11 peptides identified correctly • Critical analysis • 11/11 peptides identified correctly

  17. Cyclic Octapeptides • 30 Pure compounds • c[KHKHKWLW], c[KFQFKNWN], c[KAQAKAWA], c[KAQNKAWN] etc. • Hypothetical library (4.0  106 sequences) • Fixed K • 7 variable positions • Ala, Phe, His, Lys, Leu, Asn, Arg, Ser, Trp • Automated LCMS runs • CID of most intense ion in LC peak

  18. Cyclic Octapeptides • Thousands of sequences shortlisted • Scoring rank • 23/30 peptides identified correctly • Critical analysis • 22/30 peptides identified correctly • 11 cases where selected sequences differ • Only 5 where both are incorrect • Only 3 cases with incorrect residues

  19. Split & Pool Hexapeptides • Hexapeptides • c[KXXWLW] • X = Lys, His, Ser, Leu, Trp • 25 compounds • Split & pool synthesis on macrobeads • Randomly select 30 beads • Cleave peptide • Auto LCMS analysis • Assign sequence by inspection of spectra

  20. Split & Pool Hexapeptides • Program input • Amphipathic cyclic peptides • c[KXXZZZ] • X = Ala, Phe, Gly, His, LysLeu, Met, Asn, Arg, Ser, Thr, Val, Trp, Tyr • Z = Ala, Phe, Leu, Val, Trp • 24500 sequences • 28/30 sequences identical to the manual assignments

  21. Summary • Cyclic peptide MS sequencing software • Oriented towards split & pool library analysis • No requirement for chemical encoding • Automated • Integrates with other applications • Customizable processing

  22. Acknowledgments • Funding • The Wellcome Trust (JER) • The Skaggs Institute (KMW) • NIGMS (MRG)

  23. c[KWLWKS] • MS/MS of pure material

  24. c[KHKHLALWL] • Auto LC-MS/MS

  25. c[KSHWLW] • Split and pool single bead • Auto LC-MS/MS

  26. Compound No. Sequence No. of shortlist sequences Highest Scoring Sequence selected from CA 1 c[KWLWKE] 1055 c[KWLWKE] c[KWLWKE] 2 c[KWLWKF] 714 c[KWLWKF] c[KWLWKF] 3 c[KWLWKH] 692 c[KWLWKH] c[KWLWKH] 4 c[KWLWKS] 3733 c[KWLWKS] c[KWLWKS] 5 c[KWQWLW] 201 c[KWLWKW] c[KWLWKW] 6 c[KRWLWL] 559 c[KLWRWL] c[KRWLWL] 7 c[KEQWLW] 1055 c[KWLWKE] c[KWLWKE] 8 c[KFQWLW] 714 c[KWLWKF] c[KWLWKF] 9 c[KHQWLW] 692 c[KWLWKH] c[KWLWKH] 10 c[KKQWLW] 1221 c[KWLWKK] c[KWLWKK] 11 c[KQQWLW] 1221 c[KWLWKK] c[KWLWKK] Hexamer Sequences

  27. Compound No. Sequence No. of shortlist Sequences Highest Scoring Sequence Sequence selected by CA Rank of actual sequence 12 c[KHKHKWLW] 7145 c[KWLWKHKH] c[KWLWKHKH] 1 13 c[KHQHKWLW] 7145 c[KWLWKHKH] c[KWLWKHKH] 1 14 c[KFQFKNWN] 12174 c[KNWNKFKF] c[KNWNKFKF] 1 15 c[KNQNKFWF] 12174 c[KNKNKFWF] c[KNKNKFWF] 1 16 c[KAQAKAWA] 3041 c[KAWAKAKA] c[KAWAKAKA] 1 17 c[KAQNKAWN] 13707 c[KNWAKNKA] c[KWNAKNKA] 1 18 c[KNQNKLWL] 18720 c[KNKNKLWL] c[KNKNKLWL] 1 19 c[KHKLALWL] 14298 c[KLWLALKH] c[KLWLALKH] 1 20 c[KSKLRLRL] 17632 c[KFLFLFKA] c[KFAKFLFL] 2 21 c[KSKLFLFL] 21058 c[KSKLFLFL] c[KSKLFLFL] 1 22 c[KHQHKLWL] 14066 c[KLWLKHKH] c[KLWLKHKH] 1 Octamer Sequences

  28. 23 c[KHSHKWLW] 10322 c[KWLWKHSH] c[KWLWKHSH] 1 24 c[KSSSKWLW] 17783 c[KWLWKSSS] c[KWLWKSSS] 1 25 c[KSKSKWLW] 19611 c[KWLWKSKS] c[KWLWKSKS] 1 26 c[KSQSKWLW] 19611 c[KWLWKSKS] c[KWLWKSKS] 1 27 c[KSKWLWLW] 7204 c[KWLWLWKS] c[KWLWLWKS] 1 28 c[KLWLWLWL] 7007 c[KLWLWLWL] c[KLWLWLWL] 1 29 c[KKWLAALW] 16442 c[KKWLAALW] c[KKWLAALW] 1 30 c[KKWLWLWL] 4772 c[KWLKWLWL] c[KWLKWLWL] 7 31 c[KSKLWLWL] 11898 c[KSKLWLWL] c[KSKLWLWL] 1 32 c[KQRWLWLW] 2351 c[KWLWLWKR] c[KRWKLWLW] 23 33 c[KHKHFLWL] 12715 c[KHFKHKFH] c[KHFLWLKH] 13 Octamer Sequences

  29. 34 c[KSSKWLLW] 17899 c[KSSKWLLW] c[KSSKWLLW] 1 35 c[KKKWLWLW] 3579 c[KWLWLWKK] c[KWLWLWKK] 1 36 c[RHKKLWLW] 6757 c[KLWRHKWL] c[KKLWLWRH] 28 37 c[RHKHRWLW] 1325 c[KHRWLWRH] c[KHHRWLWR] 1 38 c[RSKKLWLW] 11089 c[KRWRKKSK] c[KWRKRKSK] >30 39 c[RSKSRWLW] 11871 c[KSRWLWRS] c[KRWLWRSS] 1 40 c[KQKKLWLW] 10764 c[KWLLWKKK] c[KKWLWKLK] 21 41 c[KWKWKWLW] 387 c[KWLWKWKW] c[KWLWKWKW] 1 Octamer Sequences

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