3.9 Trypsin Digestion
for Mass Spectrometry
1. Prepare trypsin solution:
(a) Resuspend 20 μg trypsin in 40 μL of 20 mM Tris–HCl
pH 8.
(b) For “N” number of samples, combine a total of
(N + 2) Â 2 μL trypsin and (N + 2) Â 100 μL of 50 mM
ABC in a fresh 1.5 mL microcentrifuge tube.
2. Centrifuge solution with beads (from Subheading 3.8, step
14) for 30 s at 500 Â g. Remove supernatant.
3. Add 100 μL of trypsin solution to each sample and incubate for
4 h with rotation at 37
C.
4. Add fresh 1 μg trypsin (2 μL suspended in Tris–HCl pH 8) to
each sample.
5. Incubate overnight with rotation at 37
C.
6. Next day, centrifuge beads for 2 min at 500 Â g and retrieve
100 μL supernatant with digested peptides into a fresh 1.5 mL
microcentrifuge tube; avoid beads.
7. Add 100 μL of MS H 2 O to the beads and wash by inversion
4–5 times.
8. Centrifuge for 2 min at 500 Â g and transfer supernatant to the
1.5 mL microcentrifuge tube with previously digested peptides
in step 6.
9. Add formic acid to peptides using glass pipettes for a final
concentration of 2% formic acid.
10. Spin for 5 min at 16,000 Â g, then transfer exactly 230 μL to a
fresh 1.5 mL microcentrifuge tube.
11. Speed vacuum samples just to dryness without heat (see Note
33).
12. Store dried peptides at À80
C until ready for mass spectrometry analysis [13].
4 Notes
1. For BirA* in Arabidopsis, we used 2 mM Biotin for 24 h.
Zhang et al. recommended 200 μM for 12 h, Mair et al.
recommend 50 μM biotin for 0.5 and/or 3 h, and Kim et al.
recommend 50 μM biotin for 3 h with TurboID in
Arabidopsis.
2. For example, see TakaraBio In-Fusion Cloning HD Cloning
Plus kit (TakaraBio #638909) manual for instructions on how
to design primers for single PCR fragment insert or multiple
PCR fragment inserts into the vector. Alternatively, overlapping PCR can be used to amplify a full-length product of gene
of interest with BirA* and amplify this full-length product
Biotin-Based Proximity Labeling of Protein Complexes in Planta
437
for Mass Spectrometry
1. Prepare trypsin solution:
(a) Resuspend 20 μg trypsin in 40 μL of 20 mM Tris–HCl
pH 8.
(b) For “N” number of samples, combine a total of
(N + 2) Â 2 μL trypsin and (N + 2) Â 100 μL of 50 mM
ABC in a fresh 1.5 mL microcentrifuge tube.
2. Centrifuge solution with beads (from Subheading 3.8, step
14) for 30 s at 500 Â g. Remove supernatant.
3. Add 100 μL of trypsin solution to each sample and incubate for
4 h with rotation at 37
C.
4. Add fresh 1 μg trypsin (2 μL suspended in Tris–HCl pH 8) to
each sample.
5. Incubate overnight with rotation at 37
C.
6. Next day, centrifuge beads for 2 min at 500 Â g and retrieve
100 μL supernatant with digested peptides into a fresh 1.5 mL
microcentrifuge tube; avoid beads.
7. Add 100 μL of MS H 2 O to the beads and wash by inversion
4–5 times.
8. Centrifuge for 2 min at 500 Â g and transfer supernatant to the
1.5 mL microcentrifuge tube with previously digested peptides
in step 6.
9. Add formic acid to peptides using glass pipettes for a final
concentration of 2% formic acid.
10. Spin for 5 min at 16,000 Â g, then transfer exactly 230 μL to a
fresh 1.5 mL microcentrifuge tube.
11. Speed vacuum samples just to dryness without heat (see Note
33).
12. Store dried peptides at À80
C until ready for mass spectrometry analysis [13].
4 Notes
1. For BirA* in Arabidopsis, we used 2 mM Biotin for 24 h.
Zhang et al. recommended 200 μM for 12 h, Mair et al.
recommend 50 μM biotin for 0.5 and/or 3 h, and Kim et al.
recommend 50 μM biotin for 3 h with TurboID in
Arabidopsis.
2. For example, see TakaraBio In-Fusion Cloning HD Cloning
Plus kit (TakaraBio #638909) manual for instructions on how
to design primers for single PCR fragment insert or multiple
PCR fragment inserts into the vector. Alternatively, overlapping PCR can be used to amplify a full-length product of gene
of interest with BirA* and amplify this full-length product
Biotin-Based Proximity Labeling of Protein Complexes in Planta
437