Selecting protein N-terminal peptides by combined fractional diagonal chromatography
- Author
- An Staes (UGent) , Francis Impens (UGent) , Petra Van Damme (UGent) , Bart Ruttens (UGent) , Marc Goethals (UGent) , Hans Demol (UGent) , Evy Timmerman (UGent) , Joël Vandekerckhove (UGent) and Kris Gevaert (UGent)
- Organization
- Project
- Bioinformatics: from nucleotids to networks (N2N)
- 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 alpha-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 similar to 5 d.
- Keywords
- PROTEOLYTIC CLEAVAGE SITES, QUANTITATIVE PROTEOMICS, DEGRADOMICS, CELL-DEATH, POSITIONAL PROTEOMICS, SUBSTRATE, STRATEGY, IDENTIFICATION, IN-VIVO, APOPTOSIS
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-1907040
- Chicago
- Staes, An, Francis Impens, Petra Van Damme, Bart Ruttens, Marc Goethals, Hans Demol, Evy Timmerman, Joël Vandekerckhove, and Kris Gevaert. 2011. “Selecting Protein N-terminal Peptides by Combined Fractional Diagonal Chromatography.” Nature Protocols 6 (8): 1130–1141.
- APA
- Staes, A., Impens, F., Van Damme, P., Ruttens, B., Goethals, M., Demol, H., Timmerman, E., et al. (2011). Selecting protein N-terminal peptides by combined fractional diagonal chromatography. NATURE PROTOCOLS, 6(8), 1130–1141.
- Vancouver
- 1.Staes A, Impens F, Van Damme P, Ruttens B, Goethals M, Demol H, et al. Selecting protein N-terminal peptides by combined fractional diagonal chromatography. NATURE PROTOCOLS. 2011;6(8):1130–41.
- MLA
- Staes, An, Francis Impens, Petra Van Damme, et al. “Selecting Protein N-terminal Peptides by Combined Fractional Diagonal Chromatography.” NATURE PROTOCOLS 6.8 (2011): 1130–1141. Print.
@article{1907040,
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 alpha-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 similar to 5 d.},
author = {Staes, An and Impens, Francis and Van Damme, Petra and Ruttens, Bart and Goethals, Marc and Demol, Hans and Timmerman, Evy and Vandekerckhove, Jo{\"e}l and Gevaert, Kris},
issn = {1754-2189},
journal = {NATURE PROTOCOLS},
language = {eng},
number = {8},
pages = {1130--1141},
title = {Selecting protein N-terminal peptides by combined fractional diagonal chromatography},
url = {http://dx.doi.org/10.1038/nprot.2011.355},
volume = {6},
year = {2011},
}
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