title: Selecting protein N-terminal peptides by combined fractional diagonal chromatography.
abstract: 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.
authors.affiliation: Department of Medical Protein Research, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Staes
authors.forename: Francis
authors.initials: F
authors.lastname: Impens
authors.forename: Petra
authors.initials: P
authors.lastname: Van Damme
authors.forename: Bart
authors.initials: B
authors.lastname: Ruttens
authors.forename: Marc
authors.initials: M
authors.lastname: Goethals
authors.forename: Hans
authors.initials: H
authors.lastname: Demol
authors.forename: Evy
authors.initials: E
authors.lastname: Timmerman
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
mesh_terms: D020148:Butyric Acid / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods; D002845:Chromatography / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D004952:Esters / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011422:Propionates / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods; D013388:Succinimides / Q000737:chemistry
chemical_list: D004952:Esters; D010455:Peptides; D011422:Propionates; D011506:Proteins; D020543:Proteome; D013388:Succinimides; D020148:Butyric Acid; C029658:propionic acid; C001426:N-hydroxysuccinimide