255Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_16, © Springer Science+Business Media New York 2014
Chapter 16
Preparation of Arabidopsis thaliana Seedling Proteomes
for Identifying Metacaspase Substrates by N-terminal
COFRADIC
Liana Tsiatsiani , Simon Stael , Petra Van Damme ,
Frank Van Breusegem , and Kris Gevaert
Abstract
Proteome-wide discovery of in vivo metacaspase substrates can be obtained by positional proteomics
approaches such as N-terminal COFRADIC, for example by comparing the N-terminal proteomes (or
N-terminomes) of wild-type plants to transgenic plants not expressing a given metacaspase. In this chapter
we describe a protocol for the preparation of plant tissue proteomes, including differential isotopic label-
ling allowing for a comparison of in vivo N-terminomes that serves as the starting point for N-terminal
COFRADIC studies.
Key words Metacaspases , Positional proteomics , N-terminal COFRADIC , Protease substrates ,
Neo-N- termini , Tissue samples , Degradomics1 Introduction
Identifi cation of protease substrates and characterization of prote-
ase substrate specifi cities in utmost detail rely nowadays mostly on
mass spectrometry driven proteomics (recently reviewed in [ 1 ]).
When sampling whole proteomes, protease substrates are identi-
fi ed either based on their altered mobility during gel electrophore-
sis (e.g., the PROTOMAP technology introduced by the Cravatt
lab [ 2 ]) or by exploiting the chemical reactivity of the alpha-amino
groups that, amongst others, are introduced when proteases cleave
their substrates and are referred to as neo-N-termini. The latter
technologies involve the enzymatic or chemical labelling of these
reactive groups with affi nity tags (e.g., the subtiligase approach
[ 3 ]), scavenging all other peptides on solid supports (e.g., the
TAILS approach [ 4 ]) or depleting these non N-terminal peptides
using consecutive chromatography steps. This last approach was
introduced in 2003 and termed N-terminal COmbined FRActional