Selecting protein N-terminal peptides by combined fractional diagonal chromatography

Nat Protoc. 2011 Jul 14;6(8):1130-41. doi: 10.1038/nprot.2011.355.


In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Butyric Acid / chemistry
  • Chemical Fractionation / methods
  • Chromatography / methods*
  • Chromatography, Liquid / methods
  • Esters / chemistry
  • Humans
  • Jurkat Cells
  • Mass Spectrometry / methods
  • Peptides / chemistry
  • Propionates / chemistry
  • Proteins / chemistry*
  • Proteome
  • Proteomics / methods
  • Succinimides / chemistry


  • Esters
  • Peptides
  • Propionates
  • Proteins
  • Proteome
  • Succinimides
  • Butyric Acid
  • propionic acid
  • N-hydroxysuccinimide