258
- Tris(2-carboxyethyl)phosphine (TCEP) stock solution:
150 mM TCEP ( see Note 1 ). - Iodoacetamide stock solution: 300 mM iodoacetamide.
- NAP™-10 columns (GE Healthcare Life Sciences) or similar.
- Light and heavy labelling solutions: freshly prepare 10 mM of
N -hydroxysuccinimide esters of either^12 C 4 -butyric acid (light)
or^13 C 4 -butyric acid (heavy) in 50 % of acetonitrile ( see Note 2 ). - 1 M glycine.
- Hydroxylamine.
- 10 mM ammonium bicarbonate (pH 8). Prepare fresh.
- Trypsin (e.g., sequencing-grade modifi ed trypsin from Promega).
3 Methods
- Collect seedling tissue from the two plant lines studied (e.g.,
wild-type plants versus transgenic plants that do not express a
particular metacaspase), freeze in liquid nitrogen and grind
into a fi ne powder with mortar and pestle ( see Note 3 ). - Resuspend 0.5 g of frozen ground tissue in 1 mL of pre-
chilled (4 °C) proteome extraction buffer and allow to thaw
( see Note 4 ). Transfer to a fresh tube. - Centrifuge the sample for 10 min at 16,000 × g at 4 °C, care-
fully aspirate the supernatant transfer to a fresh tube and dis-
card the pellet. - Repeat the previous step to remove any remaining debris.
- Measure the protein concentration and add solid guanidinium
hydrochloride to the cleared lysate to a fi nal concentration of
4 M ( see Note 5 ). - Add TCEP stock solution and iodoacetamide stock solution
to the protein mixtures to obtain fi nal concentrations of 15
and 30 mM, respectively. - Incubate for 30 min at 37 °C in the dark.
- Desalt the protein mixtures over, e.g., NAP™-10 columns in
1.5 mL of 1.4 M of guanidinium hydrochloride in 50 mM
sodium phosphate buffer at pH 7.5. - Add 50 μL of either light (e.g., to the seedling proteome prep-
aration of wild-type plants) or heavy (e.g., to the seedling pro-
teome preparation of metacaspase knockout plant) labelling
solution to the desalted protein mixtures. - Incubate for 1 h at 30 °C.
2.2 Preparation
of Proteomes for
Differential N-Terminal
COFRADIC Analysis
3.1 Proteome
Extraction from
Arabidopsis thaliana
Seedlings
3.2 Preparation
of Proteomes for
Differential N-Terminal
COFRADIC Analysis
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