259- Repeat steps 4 and 5 ( see Note 6 ).
- Add 1.25 μL of 1 M glycine to quench the non-reacted
N -hydroxysuccinimide esters. - Incubate for 10 min at room temperature.
- Add 10 μL of hydroxylamine ( see Note 7 ).
- Incubate for 15 min at room temperature.
- Reduce the volume of the solution to 1 mL by centrifugal
vacuum drying. - Desalt the protein mixtures over, e.g., NAP™-10 columns in
1.5 mL of 10 mM freshly prepared ammonium bicarbonate
(pH 8). - Measure the protein concentration of both samples and mix
equal amounts of samples. - Incubate this protein mixture for 10 min at 95 °C and transfer
to ice for 10 min incubation. - Add trypsin to an enzyme/substrate ratio of 1/50 (w/w).
- Incubate overnight at 37 °C.
- Centrifuge the peptide mixture for 10 min at 16,000 × g , col-
lect the supernatant, vacuum dry and store at −20 °C until
further use ( see Note 8 ).
4 Notes
- TCEP is available as TCEP·HCl, and dissolving this product
releases HCl which causes a drop in pH. Therefore, the pH of
the TCEP stock solution must be raised to pH 7.5. Typically,
500 μl of TCEP stock solution requires adding 30 μl of 5 M
NaOH to obtain a pH of 7.5. - These labelling solutions allow for incorporation of differen-
tial isotopes in the proteomes to be analyzed. The protocol for
synthesizing N -hydroxysuccinimide esters of butyric acid vari-
ants has been published [ 15 ], though note that other
N -hydroxysuccinimide esters are commercially available (e.g.,
those of acetate and trideutero-acetate). - Note that seeds of Arabidopsis thaliana Columbia (Col-0)
were overnight gas sterilized with HCl and NaOCl, then
sowed on half strength Murashige and Skoog (MS) media
plates containing 0.8 % agar and 1 % sucrose. Two plates were
sown per plant line. The plates were kept at 4 °C in the dark for
seed stratifi cation and after 3 days, were transferred to 21 °C
with a 16 h light–8 h dark photoperiod, light intensity of
80–100 μmol/m^2 /s and 70 % humidity. The duration of seed-
ling growth at 21 °C varies and depends on the expression
Preparation of Arabidopsis thaliana Seedling Proteomes for Metacaspase Degradomics