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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.

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Figure 1: Schematic workflow of the N-terminal COFRADIC procedure.
Figure 2: Examples of primary and secondary COFRADIC RP-HPLC runs.

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Acknowledgements

F.I. and P.V.D. are Postdoctoral Research Fellows of the Fund for Scientific Research (FWO)-Flanders (Belgium). This work was further supported by research grants from the Fund for Scientific Research-Flanders (Belgium; project numbers G.0048.08 and G.0440.10), the Concerted Research Actions (project BOF07/GOA/012) from Ghent University and the Interuniversity Attraction Poles (IUAP06).

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B.R. synthesized the NHS esters. A.S., F.I., P.V.D., M.G., H.D. and E.T. performed the experiments. J.V. and K.G. supervised the work. A.S., F.I., P.V.D., B.R. and K.G. wrote the paper.

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Correspondence to Kris Gevaert.

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The authors declare no competing financial interests.

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Supplementary Figure 1

Purification of pGAPase, analysis by LC-MS. (DOC 1393 kb)

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Staes, A., Impens, F., Van Damme, P. et al. Selecting protein N-terminal peptides by combined fractional diagonal chromatography. Nat Protoc 6, 1130–1141 (2011). https://doi.org/10.1038/nprot.2011.355

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