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Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage
Author(s) -
Ebhardt H. Alexander,
Nan Jie,
Chaulk Steven G.,
Fahlman Richard P.,
Aebersold Ruedi
Publication year - 2014
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7069
Subject(s) - chemistry , peptide , amino acid , peptide sequence , electrospray ionization , tandem mass spectrometry , electron transfer dissociation , enzyme , n terminus , fragmentation (computing) , biochemistry , stereochemistry , mass spectrometry , chromatography , computer science , gene , operating system
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. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.