Dimethyl isotope labeling assisted de novo peptide sequencing

J Am Soc Mass Spectrom. 2010 Dec;21(12):1957-65. doi: 10.1016/j.jasms.2010.08.007. Epub 2010 Aug 21.


Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Ascorbate Peroxidases
  • Carbon Isotopes / analysis
  • Carbon Isotopes / chemistry
  • Cattle
  • Isotope Labeling / methods*
  • Metalloendopeptidases / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism
  • Peroxidases / chemistry
  • Peroxidases / metabolism
  • Persea / enzymology
  • Plant Proteins / chemistry
  • Plant Proteins / metabolism
  • Sequence Alignment / methods*
  • Sequence Analysis, Protein / methods*
  • Serum Albumin, Bovine / chemistry
  • Serum Albumin, Bovine / metabolism
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • Carbon Isotopes
  • Peptide Fragments
  • Plant Proteins
  • Serum Albumin, Bovine
  • Peroxidases
  • Ascorbate Peroxidases
  • Metalloendopeptidases