SideChain Losses in Electron Capture Dissociation to Improve Peptide Identification

1
Side-Chain Losses in Electron Capture
Dissociation to Improve Peptide Identification

• Mikhail M. Savitski, Michael L. Nielson, and
  Roman A. Zubarev
• Laboratory for Biological and Medical Mass
  Spectrometry, Uppsala University
• Presented By Chris Keller

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Objective

• Test the extent of side chain losses in
  conventional ECD of tryptic peptides in order
  to observe
• Dependence upon Amino Acid Type and Position
• Xle Identification in High-Throughput Analysis
• Distant Side Chain Losses
• Detection of the Presence of Met
• Radical Migration
• Intracomplex H Transfer

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Introduction

• ECD
• Fourier Transform Ion Cyclotron Resonance Mass
  Spec
• Introduction of low energy electrons to trap gas
  phase ions
• Energy (lt0.2-eV)

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Equipment and Procedure

• 7 T LTQ FT mass Spec. (Thermo)
• Consecutive ECD (MS/MS)
• Computer-assisted design (CAD)
• Nano-LC system (Agilent 1100)
• Analytical Column
• Mascot Search Engine (Matrix Sciences)

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Dependence upon Amino Acid Type and Position

• Cleavage causes radical on Ca atom
• Migration of radical causes loss of largest alkyl
  group
• Example Ile loses C2H5 rather than CH3

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Dependence upon Amino Acid Type and Position,
Contd

• Analysis only covered peptides that contained
  common amino acids found in tryptic digests
• Thus Lys, Arg, Cys and Trp were exluded
• Nearly half of z ions that started with Leu and
  Ile produced w ions
• Other residues that exhibited frequent w ion
  formation were Gln, Glu, and Met
• Freq of loss decreased as distance from cleavage
  site increased

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Xle Identification in High-Throughput Analysis

• The most valuable tool of w and u ions is the
  distinguishing between the isomeric Leu and Ile
  residues
• Using w ions, of 11,303 where Xle was then
  N-terminal amino acid, there was only about a 2.5
  that did not argee
• Using u ions (X(Xle)XX), there was only 4.5
  that did not agree in the 1464 cases

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Xle Identification in High-Throughput Analysis,
Contd
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Distant Side Chain Losses

• To determine how far away from the initial
  radical position side chains can be lost from
  residues, variance analysis was employed
• Average deviation from the average value from
  different amino acids in the same relative pos.

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Distant Side Chain Losses, Contd
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Distant Side Chain Losses, Contd
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Detection of the Presence of Met

• 2668 mass spectra were analyzed for the loss of
  CH2SCH3
• Positive match found in 24
• Match to 12 that did not have Met
• 12 false-positive
• Concluded that Met side chain loss was not a good
  indicator for Met amino acid residue

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Radical Migration

• Rate of the loss of frequency drops with the
  distance to the initial radical site was
  determined for Leu, Ile, Gln, and Glu.
• Showed near exponential decay behavior
• This is known as multistep, random-walk
  propagation
• Leu in the 3d position from N-terminus in a z
  ion
• The 15 other amino acids were placed in the
  second position and their numbers recorded.

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Radical Migration, Contd
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Intracomplex H Transfer

• Reason why w ions are not appearing more
  frequently
• Due to the intracomplex transfer of H from c to
  z
• This causes z to become an even-electron
  molecule, which means side chain loss through
  previous mechanism is not possible
• H transfer and w ion formation are competing
  processes

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Intracomplex H Transfer, Contd
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Summary

• Partial side chain losses in ECD to form u and w
  ions is more widespread than previously thought.
• Losses are abundant for Leu, Ile, Glu, Asp, Gln,
  and Met
• ECD side chain loss of Xle is fairly reliable
  when used to identify amino acids
• Shows it would be a good tool for proteomics
  sequencing
• Can account for loss up to 4 residues away from
  cleavage site, and up to 6 or more for Met
• Current investigations show that multistep,
  random-walk-type H migration, possibly along the
  backbone of protein

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References

• Savitski M, Nielson, M Zubarev, R Anal Chem
  2007, 79, 2296-2302
• Nielsen, M. L. Savitski, M. M. Zubarev, R. A.
  Mol. Cell. Proteomics 2005, 4, 835-845
• Mirgorodskaya, E. Roepstorff, P. Zubarev, R. A.
  Anal. Chem. 1999, 71, 4431-4436
• Zubarev, R. A. Kelleher, N. L. McLafferty, F.
  W. J. Am. Chem. Soc. 1998, 120, 3265-3266
• Savitski, M. M. Kjeldsen, F. Nielsen, M. L.
  Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2007,
  18 (1), 113-120