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Enzymatic generatio...
Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage
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- Ebhardt, H Alexander (författare)
- ETH Zürich
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- Nan, Jie (författare)
- Lund University,Lunds universitet,MAX IV-laboratoriet,MAX IV Laboratory
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- Chaulk, Steven G (författare)
- University of Alberta
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- Fahlman, Richard P (författare)
- University of Alberta
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- Aebersold, Ruedi (författare)
- ETH Zürich
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(creator_code:org_t)
- 2014-11-07
- 2014
- Engelska 9 s.
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 28:24, s. 43-2735
- Relaterad länk:
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http://dx.doi.org/10...
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https://onlinelibrar...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini.RESULTS: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue.CONCLUSIONS: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern.
Nyckelord
- Amino Acids, Basic
- Aminoacyltransferases
- Peptides
- Sequence Analysis, Protein
- Tandem Mass Spectrometry
Publikations- och innehållstyp
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- ref (ämneskategori)
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