Computer%20Assisted%20Sequencing%20of%20Cyclic%20Peptides

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Computer Assisted Sequencing of Cyclic Peptides

• James E. Redman
• Keith M. Wilcoxen
• M. Reza Ghadiri

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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

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Peptide Synthesis
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Split-and-Pool Synthesis

• Single bead - Single compound
• Macrobead resins
• 500 mm diameter
• Dispense into plates cleave peptide
• Sufficient compound for biological testing

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Peptide Identification

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

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Example

• cKWLWKS
• MS/MS of pure material

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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

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Software Outline
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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

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Scoring

• 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

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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

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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

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Excel Interface

• Sequence menu
• Parent spectrum parameters
• CID spectrum parameters
• Library composition

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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

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Cyclic Hexapeptides

• 11 Pure compounds
• cKWLWKE, cKWLWKF, cKRWLWL etc.
• Hypothetical library (1.8 ? 106 sequences)
• Fixed Lys residue
• 5 variable residues
• 18 Possibilities
• Omit Q, I
• Manually optimized MS/MS

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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

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Cyclic Octapeptides

• 30 Pure compounds
• cKHKHKWLW, cKFQFKNWN, cKAQAKAWA,
  cKAQNKAWN 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

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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

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Split Pool Hexapeptides

• Hexapeptides
• cKXXWLW
• 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

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Split Pool Hexapeptides

• Program input
• Amphipathic cyclic peptides
• cKXXZZZ
• 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

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Summary

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

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Acknowledgments

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

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cKWLWKS

• MS/MS of pure material

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cKHKHLALWL

• Auto LC-MS/MS

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cKSHWLW

• Split and pool single bead
• Auto LC-MS/MS

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Hexamer Sequences
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Octamer Sequences
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Octamer Sequences
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Octamer Sequences