Automated Fast-Bead Synthesis of Small Peptides

Transcription

1 Automated Fast-Bead Synthesis of Small Peptides Application Note 228 Joan Stevens, Ph.D., Norbert Wodke, Tim Hegeman and Kirby Reed (Gilson, Inc.) Introduction In proteomic research, the synthesis of peptides in small quantities is required for fast-and-quick assays. These assays provide possible hits that can be synthesized later at greater concentrations. The synthesis of these peptides is usually in the 1 mg range and requires hundreds of peptides to be synthesized concurrently. The application presented offers a novel yet simple automated system to accomplish this task. An automated liquid handler is equipped with 96-well filter plates attached to a vacuum system/rack. The resin is manually added to the individual wells of the filter plate. Employing the capabilities of the liquid handler, reagents, solvents and amino acids are added to the resin based on the order and volume defined by the researcher. This information is quickly accessed through a text file into the program. The vacuum capabilities, when activated by the program, withdraw the solutions through the resins present in the individual wells/rack. Materials & Methods Instrumentation and Software Quad-Z 215 Liquid Handler: equipped with 4 independent probes 444 QuadDilutor: equipped with 1.0-mL syringes Amino Acid Rack for 50-mL conical vials Reagent Rack for 700-mL reagent bottles (4 bottles/rack) Racks for filter plates (Gilson vacuum racks hold two filter plates/rack, up to 6 filter plates. Custom racks available for Whatman filter plates employing the Whatman Vacuum Manifolds) Orbital Shaker Peptide software (Gilson design) April 2005 Page 1 of

2 Accessories and Reagents Filter Plates AcroPrep Filter Plate with 0.2 µ GHP, polypropylene (Pall part #5045) Whatman Unifilter Plate, 10 µm, polypropylene (Whatman part # ) Whatman Unifilter Plate, µm, 96-well, polypropylene (Part # ) (Will require additional valves to allow N 2 to be accessed for positive pressure during coupling/decoupling steps based on filter size. May be required if larger amounts of resin are used. *Not used in this application.) Quad-Z 215 Peptide Synthesizer Features Independent reaction blocks for up to 576 parallel peptide synthesis 1 5 mg of resin ( micromole) Simple synthesis control software Four independent probes for fast reagent delivery and washing Peptide Software AA sequence imported from.txt file or spreadsheet An Excel macro is used to populate the spreadsheet for the following requirements: cycle # letter: designates amino acid number: designates well within the series of plates Figure 1. Input screen of the Peptide Program; implements a file (.txt) to determine number of cycles (length of peptide) and placement of individual amino acids (single letter) into wells (well number of plate). April 2005 Page 2 of

3 Peptide Program Logical inputs for synthesis cycle parameters deprotection: up to 2 additions with variable times washes: up to 3 wash cycles with variable times AA addition w/ or w/o activators coupling time filtering time Cleavage is performed offline Figure 2. Peptide Software setup; user-defined values for all steps: deprotection, wash, addition, coupling and filtering. Figure 3. Example of Tray Layout: reagents in the first two rack positions, amino acids in the center and 96-well filter plates (four) in the last two rack positions. April 2005 Page 3 of

4 Results The following peptides are examples of the synthesis evaluation conducted via HPLC and MS (ESI-1Q) using a Fmoc-Gly Wang resin ( mesh, 0.6 micromoles, resin swelled prior to synthesis with DMF): V-Q-A-A-I-D-Y-I-N-G* Y-Q-Y-A-I-D-Y-I-N-G* *A = Ala, N = Asn, D = Asp, Q = Gln, G = Gly, I = Ile, Y = Try, V = Val Figure 4. VQAAIDYING [MH] /DYIN [M+ACN+H] Inset: Reverse-phase HPLC of synthesized peptide, 254 nm, 50 µl injection onto RP C18, 4.6 x 100 mm, water/acn (0.1% TFA). Figure 5. YQYAIDYING [M+NH 2 +H] /H-YQY loss 791/[M+H 2 0+H] Inset: Reverse-phase HPLC of synthesized peptide, 254 nm, 50 µl injection onto RP C18, 4.6 x 100 mm, water/acn (0.1% TFA). April 2005 Page 4 of

5 Summary The peptide synthesizer offers an automated solution to the current dilemma of synthesizing many peptides in small quantities. The system employs basic established peptide chemistry (Fmoc). The system is designed for single-bead or very small amounts of resin (1 5 mg.) Filtration plates are available as standard items through several manufacturers. Agitation is not required under the conditions used in this application because the amount of resin used is very small, but is available. The Peptide Program allows unique peptides to be synthesized per well (10 20 AA/peptide). The cleavage of the peptides from the Wang resin, as mentioned previously, was done offline; however, the system is capable of doing the cleavage with the addition of a manual step: placing a microplate under the filter plate prior to exposing the resin to the cleavage reagent. The MS data correlates with the proposed synthetic peptide; however, MS/MS was not available but should be considered for all peptide verifications. Since it is based on a liquid handler platform, the system is capable of additional LH techniques when not being used as a peptide synthesizer. The system will fit into a fume hood (~150 x 75 cm). Conclusion The automated peptide synthesizer presented offers a positive solution to the requirement of producing large numbers of peptides in small quantities for libraries or customers. The system is built using off-the-shelf items that offer a great degree of flexibility. The peptide synthesizer is designed for small peptides (10 20 AA) and employs single-bead, 1 to 5 mg resin. Production of the synthetic peptide is in the micromole range. The data presented validates the synthesizer s capabilities, and the fact that it s being employed by pharmaceutical and peptide houses validates its usefulness. Gilson, Inc. World Headquarters P.O. Box , Middleton, WI USA Telephone: (1) or (1) Fax: (1) Gilson S.A.S. 19, avenue des Entrepreneurs, F VILLIERS LE BEL France Telephone: (33-1) Fax: (33-1) sales@gilson.com, service@gilson.com, training@gilson.com April 2005 Page 5 of