Synthesis and Probing of Membrane-bound Peptide Arrays
| Synthesis and Probing of Membrane-bound Peptide Arrays | |
| Ronald Frank Department of Chemical Biology, GBF (German Research Center for Biotechnology), 38124 Braunschweig, Germany Stefan Dubel Institute for Biochemistry and Biotechnology, Technical University of Braunschweig, D-38106 Braunschweig, Germany Excerpted from Protein: Protein Interactions, Second Edition Edited by Erica A. Golemis and Peter D. Adams |
| ABSTRACT |
| The following protocol describes the synthesis of short linear peptides, or peptide pools, on modified cellulose membranes, and the detection of their protein-binding partners. Peptides are synthesized from their carboxyl termini using Fmoc-amino acid derivatives. After completion of the synthesis and cleavage of all side-chain protecting groups, the peptide array on the membrane is incubated with the potential interaction partners to identify their target sequences. |
| MATERIALS |
| Buffers, Solutions, and Reagents |
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| Special Equipment |
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| METHOD |
| All volumes given below are for one standard AC-S01 paper sheet of 8 x 12 cm and must be adjusted for more sheets, or other paper qualities and sizes. Unless otherwise stated, washes and incubation steps are carried out in sealed troughs at room temperature with gentle agitation on a rocking platform. Solvents and solutions are decanted after the time indicated. During incubations and washes, the troughs are closed with a lid. |
| Stage 1: Preparative Work Generate a list of peptides to be prepared. Multiple lists can be combined on a single membrane to fill up a complete array. The peptides can be separated after synthesis by simply cutting the membrane into appropriate sections. Cutting lines between sections can be marked out on the membrane in pencil. Select the appropriate array(s) for the chosen experiment according to number, spot size, and scale required. For manual spotting, use an 8 x 12 format (spot distance, 9 mm; spot volume, 0.5 µl for array generation, 0.7 µl for elongation cycles). An array of 17 rows with 25 spots each (spot distance 4 mm, volume 0.1 µl for array generation and 0.2 µl for elongation cycles) is recommended for the ASP22. Calculate the required volumes of Fmoc-AA solutions for each derivative and cycle. Remember that a triple coupling procedure may be necessary, and that each vial should contain a minimum of 50 µl. Consider, for example, a list of peptides that requires alanine for 26 peptides at cycle 1 on a 17 x 25 array. This cycle will require 26 (peptides) x 0.2 (µl per elongation) x 3 (couplings) = 15.6 µl of Fmoc-Ala stock solution. Therefore, you will take the minimum of 70 µl of stock solution for this vial. The SPOT software can perform this calculation. Label a set of 1.5-ml microfuge tubes with derivative and cycle code (e.g., A1), and distribute the Fmoc-amino acid stock solutions according to the calculated volumes required. Snap-freeze in liquid nitrogen and store at -70°C. |
| Stage 2: Generation of the SPOT Array Mark each membrane used with a pencil label (e.g., a number or letter at the right bottom edge) for proper orientation and tracking throughout the synthesis process. For manual synthesis, mark the spot positions on the membranes with pencil dots and place the membrane in the reaction trough. For automated synthesis, fix membranes on the platform of the spot robot. Take a 100-µl aliquot of the Fmoc-βAla stock from the freezer and bring it to room temperature. Add 1 µl of BPB stock and 4 µl of DIC. Mix, and leave for 30 min. Spot aliquots (0.5 µl for 8 x 12 array, or 0.1 µl for 17 x 25 array) of this solution on all positions of the chosen array configuration. Cover the membrane with glass plates and allow the reaction to proceed for 60 min. For peptides longer than 20mers, reduce the loading of the spots by applying a mixture of the Fmoc-βAla stock and an N-acetyl-alanine stock (1:9). This will avoid molecular crowding. Wash each membrane twice in 20 ml of acetylation mix; once for 30 sec, and once for 2 min. Incubate the membranes overnight in acetylation mix. Wash each membrane in 20 ml of DMF (3 times for 10 min each). To remove Fmoc blocking groups, incubate the membrane for 5 min in 20 ml of piperidine mix. Wash each membrane in 20 ml of DMF (4 times for 10 min each). Visualize the spots by incubating each membrane in 20 ml of DMF containing 1% BPB stock. Spots should be stained only light blue! If traces of remaining piperidine on the membranes turn the liquid dark blue, renew the staining solution and continue the staining. Wash each membrane in 20 ml of methanol or ethanol (2 times for 10 min). Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C. |
| Stage 3: Assembly of Peptides on SPOTs Take the membranes from step 9 and, for manual synthesis, number the blue spot positions with a pencil (according to the peptide lists). Place the membranes in separate reaction troughs. For automated systems, fix the unmarked membranes on the platform of the synthesizer. If necessary, cutting lines should be marked in pencil. If bound protein is to be eluted from individual spot positions after probing (Valle at al. 1999; Billich et al. 2002), these should also be marked. Take the appropriate set of Fmoc-amino acid stock aliquots for cycle 1 from the freezer, bring to room temperature, and activate by adding DIC (4 µl per 100-µl vial; ~0.25 M). Incubate at room temperature for 30 min. For manual experiments, pipette aliquots of the Fmoc-amino acid solutions onto the appropriate spots on the membrane. For automated experiments, place the vials containing the activated Fmoc-AA solutions into the rack of the spotting robot and start cycle 1. Leave for 15 min. Repeat the spotting twice and allow the reaction to proceed for 2 hr (cover the membranes on the spotter with glass plates). Monitor the amino acid coupling reaction by inspection of the spot color change. Spots should turn yellow-green during this step. If some spots remain dark blue, additional applications of Fmoc-amino acid stock solution can be made.
Wash each membrane twice with 20 ml of acetylation mix (once for 30 sec, and twice for 2 min). Incubate the membranes in fresh acetylation mix for about 10 min (until all remaining blue color has disappeared). Wash each membrane in 20 ml of DMF (3 times for 2 min each). Add 20 ml of piperidine mix and incubate for 5 min. Wash each membrane in 20 ml of DMF (at least 6 times). Incubate membranes in 20 ml of DMF containing 1% BPB. Again, the spots should be stained only light blue. If traces of remaining piperidine on the membranes turn the liquid dark blue, replace the solution and continue the staining. BPB staining is charge-specific. Therefore, it does not only bind to amino-terminal amino groups. The side chains and protecting groups of the amino acids in the peptide chain can strongly influence staining intensity. The visible color of the peptides depends on the overall charge and, therefore, on the individual amino acid sequence. Wash each membrane with 20 ml of methanol or ethanol (2 times for 5 min). Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Repeat this procedure from steps 2 to 10 for successive elongation cycles. |
| Stage 4: Terminal Acetylation Synthetic peptides mimicking fragments of a longer continuous protein chain should be amino-terminally acetylated to avoid the production of an artificially charged terminus. Alternatively, special detection labels can be attached to the amino termini of peptides by spotting respective derivatives. This is useful, for example, when peptides are applied as protease substrates and the enzyme activity is followed through the change of the label upon cleavage of the peptide. We have successfully added biotin via its in situ formed HOBt-ester (normal activation procedure) or fluorescein via its isothiocyanate (FITC) dissolved in DMF. After the final amino acid elongation cycle from the protocol above, continue as follows: Incubate each membrane in 20 ml of acetylation mix for a minimum of 30 min (until all remaining blue color has disappeared). Wash the membranes in 20 ml of DMF (3 times for 2 min), and then in 20 ml of alcohol (2 times for 5 min). Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. |
| Stage 5: Side-Chain Deprotection After the peptide assembly is complete, all side-chain-protecting groups are removed from the peptides. Trifluoroacetic acid is extremely hazardous; the following procedure must be performed in a chemical fume hood! Prepare 40 ml of deprotection mix. Place the dried membrane in the reaction trough, add deprotection mix, close the trough very tightly, and incubate overnight with gentle agitation. This harsh treatment is required for complete cleavage of protecting groups (Kramer et al. 1999; Zander 2004). Cellulose membranes less resistant than AC-S will not survive this treatment!) Subject each membrane to the following series of washes: 20 ml of DCM (4 times for 5 min each) 20 ml of DMF (3 times for 5 min each) 20 ml of alcohol (3 times for 5 min each) 20 ml of acetic acid (1 M in water) (3 times for 5 min each) This is to remove the Boc group from tryptophan residues. Wash each membrane with 20 ml of alcohol (3 times). Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C, or process further as described in the next section. |
| Stage 6: Protein-binding Assay |
| This basic procedure has been optimized for use with AP-conjugated detection antibody and a color signal development. As mentioned above, horseradish peroxidase-labeled detection agents require the use of hydrogen peroxide, which gradually destroys the peptides on the array. More sensitive detection can be achieved with a chemiluminescent substrate of AP (e.g., Immun-Star). If such a substrate is used, follow the manufacturer's instructions for steps 9 to 11 of Method A. Alternatively, test proteins can be labeled prior to incubation with the peptide array by chemical biotinylation and subsequently detected using AP-conjugated streptavidin (under the same conditions as for AP secondary antibody). If fluorescent or radioactive labeled reagents are used, adapt steps 5 to 11 of Method A accordingly. As an easy alternative to chemical labeling, in vitro coupled transcription/translation systems (TNT, Promega) can be recommended (Niebuhr et al. 1997). Important: If using Method A, prior to probing your protein with the peptide spots on the membrane, always do a "pre-run" in which you first apply this protocol while omitting steps 5 and 6. This is necessary to control for unspecific signals from components of the detection process or remaining proteins from a previous experiment on the same membrane. However, in case the proteins are electro-transferred and detected on a secondary nitrocellulose membrane (Method B, below), this precaution does not apply. Method A Place a single membrane in a polystyrene plate, and wet it with a few drops of methanol or ethanol. This is to enhance rehydration of any peptide spots that might be hydrophobic. The peptide locations should not be visible as white spots! If this happens, extend the alcohol treatment in a sonication bath at room temperature until spots have disappeared Wash the membrane in 10 ml of TBS (3 times for 10 min each). Incubate overnight in 10 ml of MBS. The blocking conditions can be critical to the success of an experiment, and, depending on the protein of interest, it may be necessary to try a number of different blocking solutions to optimize the signal-to-noise ratio. The following solutions represent increasingly stringent blocking conditions: (i) 2% (w/v) skim milk powder in TBS, (ii) 2% (w/v) skim milk powder, 0.2% (v/v) Tween-20 in TBS, (iii) MBS, (iv) MBS with 50% (v/v) horse serum. In our hands, blocking solution iii works best for most applications. Wash the membrane once in 10 ml of T-TBS for 10 min Incubate for 2-4 hr in the presence of probe antibody (or protein) diluted in 8-10 ml of MBS. For monoclonal antibodies, or pure proteins, use ~4-5 µg of purified antibody per milliliter of incubation volume. When using a polyclonal serum, we recommend a dilution of 1:100. It is not necessary to use a large volume of protein solution for the incubation. However, make sure that the membrane is completely covered throughout the incubation. To prevent drying out, use a lid, or seal the membrane in a plastic bag. Wash the membrane 3 times in 10 ml of T-TBS (for 10 min each). Incubate for 1-2 hr with AP-conjugated secondary antibody, diluted in 10 ml of MBS. Wash the membrane 2 times in 10 ml of T-TBS (for 10 min each). Wash the membrane 2 times in 10 ml of CBS |
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