6. Option C: Purified proteins can also be recovered by trypsin
digestion directly on the ANTI-FLAG
® M2 Affinity agarose
beads.
7. Wash the beads three times with 200μL of 50 mM ammonium
bicarbonate, pH8.0
8. Resuspend the beads in 40 μL of 50 mM ammonium bicarbonate before trypsin digestion.
Of note, 10% of the eluted material will be kept for quality
control purposes (gel separation and silver staining). The remaining
90% will be used for MS analysis (Fig. 1).
3.2 Peptide-Based
Purification
Peptide pulldown consists of linking a synthetic peptide to a matrix
which consists of agarose beads in general. As a PDZBM usually
corresponds to the carboxy-terminus end of the peptide mimicking
the carboxyl end of protein, it is crucial to let this side free and then
use the best strategy to cross link the beads via the N-terminus end
of the peptide. For this purpose, we recommend to use NHS
activated beads to covalently couple peptides via their primary
amino terminal group. This chemical reaction is easy to perform
and chemically stable (see Note 13).
If the synthesized peptide is composed of an internal lysine
(s) within the sequence or near the PDZBM, NHS coupling may
occur on its lateral chain and may impair the PDZBM–PDZ interaction. In this particular case, we recommend to use N-terminal
biotinylated peptides which can be coupled to streptavidin beads
(see Note 14).
3.2.1 Design of Peptide
Containing a PDZBM
1. Use a 15-mer peptide corresponding to the 15 last amino acids
of the protein which encompasses the PDZBM (see Note 15).
2. As a control, use a 12-mer peptide with the same sequence but
lacking the last tree amino acids.
3. The amino terminus should be free or biotinylated in case of
using NHS activated beads or streptavidin beads, respectively.
4. The carboxy-terminal side should be a carboxyl group
(-COOH).
5. Purity of the peptide should be above 95%.
6. Minimal quantity of peptide should be above 4mg.
3.2.2 Coupling
the Peptide to NHS Beads
All solutions should be cooled at 4
C. This protocol is adapted to
prepare 0.5 mL of peptides coupled to beads (theoretical concentration: 2 μmole peptides/mL of packed beads). A typical peptide
pulldown experiment should include at least three conditions
(Fig. 4a), namely, beads with wild-type PDZBM peptide, beads
with a mutant PDZBM peptide, and beads with no peptide.
Identification of Associated PDZ Proteins
25
digestion directly on the ANTI-FLAG
® M2 Affinity agarose
beads.
7. Wash the beads three times with 200μL of 50 mM ammonium
bicarbonate, pH8.0
8. Resuspend the beads in 40 μL of 50 mM ammonium bicarbonate before trypsin digestion.
Of note, 10% of the eluted material will be kept for quality
control purposes (gel separation and silver staining). The remaining
90% will be used for MS analysis (Fig. 1).
3.2 Peptide-Based
Purification
Peptide pulldown consists of linking a synthetic peptide to a matrix
which consists of agarose beads in general. As a PDZBM usually
corresponds to the carboxy-terminus end of the peptide mimicking
the carboxyl end of protein, it is crucial to let this side free and then
use the best strategy to cross link the beads via the N-terminus end
of the peptide. For this purpose, we recommend to use NHS
activated beads to covalently couple peptides via their primary
amino terminal group. This chemical reaction is easy to perform
and chemically stable (see Note 13).
If the synthesized peptide is composed of an internal lysine
(s) within the sequence or near the PDZBM, NHS coupling may
occur on its lateral chain and may impair the PDZBM–PDZ interaction. In this particular case, we recommend to use N-terminal
biotinylated peptides which can be coupled to streptavidin beads
(see Note 14).
3.2.1 Design of Peptide
Containing a PDZBM
1. Use a 15-mer peptide corresponding to the 15 last amino acids
of the protein which encompasses the PDZBM (see Note 15).
2. As a control, use a 12-mer peptide with the same sequence but
lacking the last tree amino acids.
3. The amino terminus should be free or biotinylated in case of
using NHS activated beads or streptavidin beads, respectively.
4. The carboxy-terminal side should be a carboxyl group
(-COOH).
5. Purity of the peptide should be above 95%.
6. Minimal quantity of peptide should be above 4mg.
3.2.2 Coupling
the Peptide to NHS Beads
All solutions should be cooled at 4
C. This protocol is adapted to
prepare 0.5 mL of peptides coupled to beads (theoretical concentration: 2 μmole peptides/mL of packed beads). A typical peptide
pulldown experiment should include at least three conditions
(Fig. 4a), namely, beads with wild-type PDZBM peptide, beads
with a mutant PDZBM peptide, and beads with no peptide.
Identification of Associated PDZ Proteins
25