257α-amino group (in vivo) or a butyrylated α-amino group (in vitro),
and non N-terminal peptides (i.e., internal peptides and C-terminal
peptides) carrying a primary α-amino group. Strong cation
exchange chromatography (SCX), when performed at acidic pH
(pH < 3) enriches for N-terminal peptides as well as for C-terminal
peptides devoid of basic amino acids [ 9 ]. Inevitably, some internal
peptides are co-enriched by this SCX step, but they are later
removed together with the C-terminal peptides by the actual
COFRADIC step. Now, peptides are separated by reverse-phase
chromatography (RP-HPLC) in a distinct number of fractions.
Peptides in these fractions are treated with 2,4,6-trinitrobenzene
sulfonic acid (TNBS) which reacts highly with primary amino
groups and attaches a very hydrophobic trinitrophenyl group onto
internal peptides and C-terminal peptides. Following a series of
identical RP-HPLC separations, in each initial peptide fraction, the
N-terminal peptides are separated from the modifi ed, more hydro-
phobic internal and C-terminal peptides and in this way isolated,
enriched and ready for subsequent analysis by mass spectrometry.
Many of the protease degradomics studies done so far with the
N-terminal COFRADIC method used mammalian cell cultures that
are readily metabolically labelled (e.g., using isotopic variants of
essential amino acids (SILAC) [ 10 ]) and thus allow for a direct
comparison of two or more N-terminal proteomes (e.g., [ 11 ]).
Although plant proteins can also be metabolically labelled for subse-
quent proteome studies (e.g., using nitrogen-15 enriched nitrogen
salts [ 12 ]), post-identifi cation analysis of data can be cumbersome
and needs specifi c data analysis software tools (e.g., [ 13 ]).
In this chapter, we describe a protocol for preparing plant
proteomes—here described for Arabidopsis seedling proteomes,
but generally applicable to other plant tissues as well—including
post-metabolic labelling events that allow for a direct comparison
of the N-terminomes of two different plant tissue proteomes. When
comparing proteomes of wild-type plants with plants not express-
ing a given metacaspase (or any other protease), using the whole
procedure, N-terminal peptides indicative for substrate processing
by this metacaspase in wild-type plants, can be identifi ed [ 14 ].2 Materials
- Proteome extraction buffer: 1 % (w/v) 3-((3-cholamidopro-
pyl)dimethylammonio)-1-propanesulfonic acid (CHAPS),
0.5 % (w/v) deoxycholate, 0.1 % (w/v) SDS, 5 mM ethylene-
diaminetetraacetic acid (EDTA), and 10 % glycerol in
phosphate- buffered saline (PBS) (pH 7.5). Add the suggested
amount of a mixture of protease inhibitors according to the
manufacturer’s instructions (e.g., Complete Protease Inhibitor
Cocktail Tablets from Roche Applied Science). - Guanidinium hydrochloride.
2.1 Proteome
Extraction from
Arabidopsis thaliana
Seedlings
Preparation of Arabidopsis thaliana Seedling Proteomes for Metacaspase Degradomics