Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage
(2014) In Rapid Communications in Mass Spectrometry 28(24). p.43-2735- Abstract
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... (More)
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.
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- author
- Ebhardt, H Alexander ; Nan, Jie LU ; Chaulk, Steven G ; Fahlman, Richard P and Aebersold, Ruedi
- organization
- publishing date
- 2014-12-30
- type
- Contribution to journal
- publication status
- published
- keywords
- Amino Acids, Basic, Aminoacyltransferases, Peptides, Sequence Analysis, Protein, Tandem Mass Spectrometry
- in
- Rapid Communications in Mass Spectrometry
- volume
- 28
- issue
- 24
- pages
- 9 pages
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- scopus:84954445952
- pmid:25380496
- ISSN
- 1097-0231
- DOI
- 10.1002/rcm.7069
- language
- English
- LU publication?
- yes
- id
- bc76bb03-e834-4ba6-abdb-afe1a0776e81
- date added to LUP
- 2016-09-07 22:49:37
- date last changed
- 2025-01-12 10:59:06
@article{bc76bb03-e834-4ba6-abdb-afe1a0776e81, abstract = {{<p>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.</p><p>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.</p><p>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.</p><p>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.</p>}}, author = {{Ebhardt, H Alexander and Nan, Jie and Chaulk, Steven G and Fahlman, Richard P and Aebersold, Ruedi}}, issn = {{1097-0231}}, keywords = {{Amino Acids, Basic; Aminoacyltransferases; Peptides; Sequence Analysis, Protein; Tandem Mass Spectrometry}}, language = {{eng}}, month = {{12}}, number = {{24}}, pages = {{43--2735}}, publisher = {{John Wiley & Sons Inc.}}, series = {{Rapid Communications in Mass Spectrometry}}, title = {{Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage}}, url = {{http://dx.doi.org/10.1002/rcm.7069}}, doi = {{10.1002/rcm.7069}}, volume = {{28}}, year = {{2014}}, }