De Novo Sequencing of Wolf Spider Antimicrobial Peptides

1
De Novo Sequencing of Wolf Spider Antimicrobial
Peptides

• Ref J. Mass Spectrom. 2004 39 193201
• Presented by Michael Armani

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Outline

• Background and Purpose
• Procedure for Peptide Isolation
• Detection of Antimicrobial Activity
• Uses of mass spectrometry
• De Novo Sequencing methods
• Results of MS
• Conclusion

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What are antimicrobial peptides?

• The key elements in an animals or plants immunity
  against bacteria and fungi
• Inhibit growth
• Gram-positive
• Gram-negative
• Fungi

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Two types of linear cationic AMPS

• From arthropods
• 13-27 amino acids
• Cecropin-like from house fly
• Mellitin-like from bee,spider
• We will look at lycocitin, from the wolf spider

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Why determine the sequence?

• Homology studies
• Predictions of structure/function
• To enable synthesis or detection
• Verify new experimental MS methods
• Fight growing antibiotic resistance
• Add to very few detailed studies of the wolf
  spider venom

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How are lycocitins Isolated?

• Wolf spiders from Uzbekistan
• Venom glands dissected, homogenized
• Extract centrifuged, supernatant freeze-dried
• Extract eluted by SE-HPLC (figure)

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Size Exclusion HPLC

• Sections C and D selected by antimicrobial assay

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Antimicrobial assay procedure

• Distribute each fraction A-G on 96 well plate
• Add gram-positive and gram-negative bacteria
• Add fungi
• Quantify growth by light absorbance (620nm)
• Normalize results to signal from synthetic
  Lycocitin I and II

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Reverse Phase HPLC

• Add fractions C and D from previous step
• Subject to acetonitrile gradients

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Isolating Lycocitins by MS (1)

• Crude extract subject to MALDI-TOF-MS
• Significant peaks were chosen including
• 1960.49, 1988.86, 3149.75, and 16 other peaks.
• Lycocitin 1 2 and 3

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Isolating Lycocitins by MS (2)

• The active fractions CD were subject to
  ESI-TOF-MS (not published)
• Lycocitin 3 found in fraction C
• Lycocitins 1-3 found in fraction D
• Two new peptides found in fraction D that
  co-eluted with Lycocitin 3 2034.20 and 2340.28

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Determining the structure

• The amino acid was determined by N-terminal
  sequencing
• ECD-MS for c and z ions (first)
• cHO-A-HPO3(-H)
• -A-OH(-H)
• CID-MS/MS for b and y ions (second)
• HO-A-O(-H)
• O-A-OH(-H)

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Determining the structure

• Two Mass analyzers
• Hybrid quadropole TOF (mainly for CID)
• Fourier transform ICR (ultra high resolution)
• Processed by Inspector software

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ECD-MS c and z ions (Lycocitin 3)
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What about that unknown?

• Mass of 170.1 matches AlaVal (170.105 Da) and
  Leu/IleGly
• Second one determined by multiple sotage assisted
  dissociation (MSAD)
• Shows cleavage between a G and L

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CAD spectrum of y17 fragment of lycocitin 3
obtained by the MSAD technique into the hexapole
of FTICR instrument. The b15 2C fragment shows
cleavage between LeuGly amino acid pair.
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CID tandem mass data eliminates tyrosine
posibility no (136 immonium) to assign fragment
ions
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CID tandem mass data loss of 64 explained by
Mox groups on peptide 2340
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Final Results
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Conclusions

• Antimicrobial peptides were identified
• ECD combined with CID-MS/MS and MSAD can identify
  long sequences de novo
• ECD Reduces high mass of molecule
• Is redundant but precise
• Can not identify Leu from Ile

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Extra data Lycocitin 1
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Extra data Lycocitin 2