Caspases,Paracaspases, and Metacaspases Methods and Protocols

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DIagonal Chromatography (COFRADIC, [ 5 ]). More recently,

the Overall lab and our lab have published positional proteomics

approaches that enable the enrichment of protein C-terminal pep-

tides which also serve as proxies for protease substrates, and thus

yield complementary information on proteolytic events [ 6 , 7 ].

The general N-terminal COFRADIC procedure is schemati-

cally depicted in Fig. 1. Briefl y, prior to digestion of the sampled

proteins with a specifi c protease such as trypsin, all primary amino

groups in proteins—N-terminal α-amino groups and lysine ε-amino

groups—are chemically blocked, for instance by butyrylation (e.g.,

[ 8 ]). Note that by using isotopic variants of butyric acid (here), this

essential step in the overall COFRADIC procedure allows for a

direct comparison of two samples. Because of the way that lysine is

chemically modifi ed, trypsin will now only cleave at arginine resi-

dues and essentially render two types of peptides; protein N-terminal

peptides, including neo-N-terminal peptides, carrying an acetylated

Cell/tissue extraction

Reduction and alkylation of Cys residues

Acylation of protein α-amines (proteins) and ε-amines (Lys residues)

Trypsin digestion

SCX at low pH

RP-HPLC fractionation of peptides

TNBS modification of internal and C-terminal peptides

RP-HPLC isolation of N-terminal peptides

Fig. 1 Schematic workfl ow of the N-terminal COFRADIC protocol. The steps not

outlined in this chapter are shown in grey

Liana Tsiatsiani et al.

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