combinatorial peptide library protocols

Synthesis of a One-Bead One-Compound Combinatorial Peptide Library Kit S. Lam and Michal Lebl 1. Introduction The four general methods to generate and screen a huge combinatorial pep- tide library +-lo7 peptides) are: biological libraries such as filamentous phage (I), plasmid (2)) or polysome (3) libraries; the “one-bead one-compound” syn- thetic combmatonal library method or the “Selectlde process” (4-6); synthetic peptide library methods that require deconvolution, such as an iterative approach (7,8), positional scanning (9); orthogonal partition approach (JO), or recurse deconvolution (II); and synthetic library using affinity column selection method (12,13). There are advantages and disadvantages m each of these methods. In gen- eral, the main advantages of the biological library method are that large pep- tides can be displayed on a filamentous phage library, and that large protein folds can be mcorporated into the library. However, the main disadvantage is that biological libraries, in general, are restricted to all L-amino acids. In con- trast, the remaining three methods all use synthetic libraries; therefore, o-amino acids, unnatural ammo acids, nonpeptide components, and small rigid scaffoldings can all be incorporated into these libraries. The “one-bead one-compound” library is based on the concept (4,5) that when a solid-phase split synthesis method (4,8,14) is used, each solid-phase particle (bead) displays only one peptide entity although there are approx 1013 copies of the same peptide in the same bead. The resulting peptide-bead library (e.g., lo7 beads) is then screened in parallel using either “on-bead” binding assays (15) or “solution phase-releasable” assays (16) to identify peptide-beads with the desired biologic, biochemical, chemical, or physical properties. The From Methods m Molecular Biology, vol 87 Combmatonal Peptrde Library Protocols Edlted by S CablIly 0 Humana Press Inc , Totowa, NJ 2 Lam and Lebl positive peptide-beads are then physically isolated for microsequencing with an automatic protein sequencer. In this chapter, detailed methods for the syn- thesis of a random “one-bead one-compound” combinatorial peptide library will be described. Chapters 2 and 10 give examples of two general screening methods for such libraries. 2. Materials 2.1. Chemicals 1. Tenta-Gel Resin S-NH, (90-100 pm) resin may be obtained from Rapp Polymere, Tubmgen, Germany (see Note 1). 2. Fmoc amino acids with standard side chain-protectmg groups, N-hydroxy- benzotriazole (HOBt), benzotriazolyl-oxy-trisdimethylammo-phosphonmm hexafluorophosphate (BOP), diisopropylethylamme (DIEA), diisopropyl- carbodumide (DIC), piperidme, trifluoroacetic acid (TFA), nmhydrm, may be obtained from many different suppliers, such as Bachem (Torrance, CA), Bio- science (King of Prussia, PA), Advanced ChemTech (Louisville, KY), Novabiochem (San Diego, CA), and Peptides International (Louisville, KY) 3. Technical grade solvents such as dimethylformamide (DMF) or dichloromethane (DCM) may be obtained from many different chemical suppliers HPLC-grade DMF for the coupling may be obtamed from Burdock and Jackson, Muskegon, MI. Ethanol, phenol, p-cresole, thioamsole, ethanedithiol, pyndme, and potas- sium cyanide may be obtained from many different chemical suppliers. 4 0 1 g/mL Nmhydrm in ethanol 5 4 g/mL Phenol m ethanol. 6 10 mM Potassmm cyanide, stock solution. 7. 50% Piperidme m DMF 8. Reagent K: TFAlp-cresolelwaterlthioamsole/ethanedithiol, 82 5*5:5:5*2.5. (v/v/ v/v/v) 9 10% DIEA m DMF. 10 Dimethylsulfoxide (DMSO)/Amsole/TFA, 10:5:85 2.2. Apparatus 1. Polypropylene vials (5-lo-mL) may be purchased from Baxter Scientific Prod- ucts, McGaw Park, IL. Polyethylene disposable transfer pipets may be purchased from Elkay Products, Shrewsbury, MA. 2 Motorized rockmg platform. 3 Randomization glass vessel (chromatography column 5-6 x 18 cm) fitted with a medmm glass smtered frit connected to vacuum and nitrogen via a two-way valve from below The three positions of the valve are “off,” “vacuum,” or “nitrogen.” 4 Recnculatmg water aspirator or a solvent-resistant vacuum pump with cold trap 5 Nitrogen tank. One-Bead One-Compound 3. Methods 3.1. Synthesis of a Linear Pentapeptide Library As indicated earlier, a solid-phase split synthesis method (4,8,14) is used to generate a random peptide library. The composition and final structure of the peptide library depends on the number of amino acids (one or more) used m each coupling cycle and the number of coupling cycles used. The final peptide library may be linear or cyclic, or have specific secondary structures. For sim- plicity, the method for the synthesis of a linear pentapeptide library with all 19 eukaryotic amino acids except cysteine is given below: 1. Swell 10 g TentaGel Resin S-NH, beads (- 0 25 mEq/g, see Notes 1 and 2) for at least 2 h m HPLC-grade DMF with gentle shaking in a silicomzed flask. 2 Wash the beads twice with HPLC-grade DMF in the slllcomzed randomlzatlon vessel as follows* add 75 mL DMF from the top, gently bubble nitrogen from below through the smtered glass for 2 min, then remove the DMF by vacuum from below (see Note 3). 3 Transfer all the beads to a slllcomzed flask in HPLC-grade DMF Then dlstrlbute the beads into 19 equal allquots. A disposable polyethylene transfer plpet IS extremely useful m the even distribution of the beads mto each polypropylene vial (see Note 4). 4 Allow the beads to settle and remove most of the DMF above the settled bead surface from each polypropylene reaction vial. 5 Add threefold molar excess of each of the 19 Fmoc-protected ammo acids (see Note 5) and threefold molar excess of HOBt to each reaction vial using a mml- ma1 volume of HPLC-grade DMF. 6. Add threefold molar excess each of BOP and DIEA to each reaction vial to ml- tlate the coupling reaction. 7. Cap the reaction vials tightly and rock them gently for 1 h at room temperature 8. To confirm the completion of couplmg reaction, plpet a minute amount of resin from each reaction vial into small borosilicate glass tubes (6 x 50-mm) and per- form ninhydrm test (17) as follows: Wash the minute quantity of resin m the small glass tubes (6 x 50-mm) sequentially with the following solvents* DMF, t-amyl alcohol (2-methylbutan-2-ol), acetic acid, t-amyl alcohol, DMF, and ether Add to each tube one drop of each of the following three reagents, (ninhydrin m etha- nol (0.1 g/mL), phenol m ethanol (4 g/mL), and potassium cyanide stock solution diluted 50 times with pyridme. Place the tubes m a heating block at 120°C for 2 min. Observe the color intensity of the beads under a microscope. To ensure complete couplmg, every bead from the minute quantity of sample beads should be nmhydrin negative, I e , straw yellow color. 4 Lam and Lebl 9 If the couplmg IS mcomplete (some beads remamed purple or brown with nmhy- drm test), remove the supernatant from those reaction vials and add fresh Fmoc- protected ammo acids, BOP, DIEA, and HOBt mto the reaction vial for addmonal coupling 10. If the couplmg 1s complete (beads remained straw yellow color with nmhydrm test) discard the supernatants of each reaction vial, and transfer and wash all the beads to the randomtzation vessel with technical grade DMF 11 After all the 19 couplmg reactrons are completed, all the beads are transferred to the randomizatton vessel Wash the beads (8 times, 2 mm each) with technical grade DMF 12 Add 75 mL 50% ptpertdme (m DMF) to the randomtzatton vessel to remove the Fmoc protectmg group After 10 mm, remove the ptpertdme and add 75 mL fresh 50% prpertdme. After another 10 mm, wash the beads 8 times wtth techmcal grade DMF and twtce with HPLC-grade DMF 13 Distribute the beads mto each of the 19 reaction vials and carry out the next couplmg reaction as described above 14. After all the randomtzatton steps are completed, remove the Fmoc protectmg group with prpertdine as described above 15 After thorough washing with technical grade DMF (5X) followed by DCM (3X), add 10 mL of reagent K (18) to the randomrzatron vessel for 3 h at room temperature 16. Wash the deprotected resms thoroughly with DCM (3X), followed by technical grade DMF (5X), then once with 10% DIEA to neutralize the resin 17. After thorough washing with technical grade DMF, store the bead library m HPLC-grade DMF at 4°C. Alternatively, the bead library can be washed thor- oughly with water and stored in 0.1 M HCl or 0.1 Mphosphate buffer with 0 05% sodmm azide. 3.2. Synthesis of a Cyclic Peptide Library The synthesis of a cyclic peptide library (disulfide bond formation) is essen- tially the same as that of the linear library except that Fmoc-Cys (Trt) is added at the carboxyl as well as amino terminus of the linear random peptide After deprotectton, add a mixture of DMSO/Anisole/TFA (see Subheading 2.1., item 10) into the resin; incubate overnight at room temperature. After thor- ough washing, store the library at 4°C as described above. 4. Notes 1 We have tested several commerctally available resins for our library synthesis The two satisfactory resins are TentaGel (polyethylene grafted polystyrene beads) and Pepsyn gel (polydimethylacrylamtde beads) Overall, the TentaGel 1s prefer- able as it is nonsticky and mechanically more stable However, unlike Pepsyn gel, the level of substrtutron of each TentaGel bead is far from uniform Wtth the advent of combmatorral chemistry, we anticipate newer resins entering the market m the near future One-Bead One-Compound 5 2. TentaGel already has a long polyethylene linker and we do not routmely add additional linker for our library synthesis In contrast, a linker (preferably a hydrophilic lmker) is necessary for the synthesis of a peptide library with polydimethylacrylamide beads. We have used Fmoc-P-alanme and/or Fmoc- aminocaprorc acid as linkers in the past. However, aminocaproic acid is rather hydrophobic A polyethyleneglycol-based amino acid (Shearwater, Polymers, Huntsville, AL) is probably preferable. 3 All glass vessels should be sdiconized thoroughly prior to use Besides using nitrogen bubbling through the randomization vessel to mix and wash the beads, we have also prepared libraries in hourglass reaction vessels (Peptides Interna- tional, Louisville, KY), usmg rocking motion to mix the resins. 4. Each polypropylene reaction vial should be engraved with a letter correspondmg to a specific amino acid to ensure no mix-up during the synthesis 5. We often omit cysteines from the synthesis of linear peptide libraries to avoid the complication of intracham and/or interchain crosslinking Acknowledgments This work was partially supported by NIH grants CA23074 and CA17094. Kit S. Lam is a scholar of the Leukemia Society of America. References 1 Scott, J K. and Smith, G. P. (1990) Searchmg for peptide ligands with an epitope library. Science 249,386-390. 2. Schatz, P. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme A 13 residue consensus peptide specifies biotinylation m Escherichia cob Biotechnology 11,1138-l 143. 3. Kawasaki, G. (199 1) Cell-free synthesis and isolation of novel genes and polypep- tides. PCT International Patent Application W09 l/05058. 4 Lam, K. S., Salmon, S. E , Hersh, E M., Hruby, V. J , Kazmierski, W. M , and Knapp, R. J. (1991) One-bead, one-peptide: a new type of synthetic peptide library for identifymg bgand-bmdmg activity. Nature 354,82-84 5 Lebl, M., Krchnak, V., Sepetov, N F., Seligmann, B., Strop, P., Felder, S and Lam, K S (1995) One-bead-one structure combmatorial libraries. Bzopolymers 37,177-198. 6 Lam, K S , Lebl, M , and Krchnak, V. (1997) The “one-bead-one-compound” combinatorial library method. Chem. Rev. 97,41 l-448 7 Geysen, H. M , Rodda, S J., and Mason, T J. (1986) A prior-z delmeation of a peptide which mimics a discontmuous antigenic determinant. Mol. Immunol. 23, 709-715. 8. Houghten, R. A , et al. (199 1) Generation and use of synthetic peptide combmato- rial libraries for basic research and drug discovery. Nature 354,84-86 9. Dooley, C. T and Houghten, R A (1993) The use of positional scanning syn- thetic peptide combinatorial libraries for the rapid determination of opioid recep- tor ligands Life Scl. 56, 1509-1517 6 Lam and Lebl 10. Deprez, B , Willard, X , Bourel, L , Coste, H , Hyafil, F., and Tartar, A (1995) Orthogonal combmatorial chemical libraries J Am. Chem. Sot. 117,5405-5408 11 Erb, E , Janda, K., and Brenner, S. (1994) Recenstve deconvolutton of combma- torial chemtcal ltbraries Proc. Nutl Acad. Scz USA 91, 11,422-l 1,425 12. Zuckermann, R. N , Kerr, J. M , Slam, M A , Banvtlle, S. C., and Santa, D V (1992) Identification of highest-affinity ligands by affinity selection from eqmmo- lar pepttde mixtures generated by robotic synthesis. Proc Natl. Acad. Scz. USA 89,4505-4509. 13 Songyang, Z., Carraway, K L , Eck, M. J , Harrtson, S C., Feldman, R. A , Mohammadi, M , Schlessmger, J , Hubbard, S. R , Smith, D P , Eng, C., Lorenzo, M. J., Ponder, B. A J , Mayer, B J , and Cantley, L. C (1995) Catalytic spectfrc- tty of protein-tyrosme kmases 1s crmcal for selecttve stgnallmg Nature 373, 536-539 14. Furka, A., Sebestyen, F., Asgedom, M., and Dtbo, G (1991) General method for rapid synthesis of multicomponent peptide mixtures. Int J Peptzde Protein Res 37,487+93. 15 Lam, K. S and Lebl, M (1994) Selectide technology-bead bmdmg screening Methods f&372-380 16 Lebl, M , Krchnak, V , Salmon, S E., and Lam, K. S (1994) Screenmg of com- pletely random one-bead-one-pepttde libraries for activmes m solution MethodA f&381-387. 17 Kaiser, E., Colescott, R. L , Bossmger, C D., and Cook, P. I. (1970) Color test for detection of free terminal ammo groups m the solid-phase synthesis of pepttdes Anal. Blochem. 34,595-602. 18 King, D. S., Fields, C G , and Fields, G. B. (1990) A cleavage method which mmtmtzes side reactions followmg Fmoc solid phase pepttde synthesis Znt. J. Peptlde Protein Res. 36,255-266. Enzyme-Linked Calorimetric Screening of a One-Bead One-Compound Combinatorial Library Kit S. Lam 1. Introduction In the “one-bead one-compound” combinatorial library method, each bead displays only one chemical compound although there are approx 1013 copies of the same compound in and on the same bead (I-3). With an appropriate detec- tion scheme, compound-beads with specific biological, physical, or chemical properties can be identified, and physically isolated, and then their chemical structure can be determined. In biological systems, one important property that is of interest is the binding property between a ligand and a ligate. The hgate or acceptor molecule could be an enzyme (4-6)) an antibody (1,7,8), a receptor (9,10), a structural protein, or even small molecules (II). Furthermore, the “one-bead one-compound” library method can also be applied to the discovery of ligands that bind to the whole viral particle, bacteria, or mammalian cell by screening for compound-beads that bind to intact cells. When we mix a ligate with an “one-bead one-compound library,” some com- pound-beads may be coated by the ligate. This interaction can be detected by either a labeled ligate or a labeled secondary probe that recognizes the ligate. Common labels are enzyme, fluorescent probe, color dye, or radionuclide. There are advantages and disadvantages to each of these methods. The choice of detection scheme depends largely on the nature and availability of specific labeled ligates. From our experience, enzyme-linked calorimetric assay is prob- ably the most convenient, economical, and rapid screening method that does not require any elaborate equipment (12). Methods for the preparation of the peptide-bead library are detailed in Chapter 7 of this volume. Details on the enzyme-linked calorimetric screening method will be given in the next sections, From Methods m Molecular Bology, vol 87 Combmatonal Pep/de Library Protocols Edlted by S CablIly 0 Humana Press Inc , Totowa, NJ 7 8 Lam 2. Materials All the reagents needed are standard enzyme-linked immunosorbent assay (ELISA) reagents and are readily available from many biochemical and chemi- cal companies. The following buffers are needed for the screening: 1. PBS-Tween. 8 mM,Na2HP04, 1.5 mMKH2P04, 137mMNaC1,2.7mMKCl,pH 7.2, with 0.1% Tween-20 (v/v) 2 Binding Buffer 16 m&Z Na2HP04, 3 mM KH,PO,, 274 mM NaCl, 5 4 mM KCl, pH 7 2, with 0.1% Tween-20 (v/v) and 0.1% gelatm (w/v). 3 TBS. 2 5 n&Z Trts-HCl, 13 7 mM NaCl, and 0 27 mM KCl, pH 8 0 4. BCIP/Alkalme phosphatase buffer. 1.65 5-Bromo-4-chloro-3-mdolyl- mg phosphate (BCIP) m 10 mL of 0 lMTris-HCl, 0 lMNaC1 with 2.34 mMMgCl,, pH 8.5-9 0 5 Gelatin. 0 1% in water 6 6M Guamdme HCl, pH 1 0 3. Methods 3.1. Screening with an Enzyme-Linked Ligate Common enzymes used in ELISA are alkaline phosphatase, horseradish per- oxrdase, P-galactosrdase, and glucose oxidase. From our experience the alka- line phosphatase system is more specific and tends to produce the least artifact when we screen a “one-bead one-compound” library. 1 If ligate-alkaline phosphatase complex is not commercially available, one may conmgate the ligate to alkaline phosphatase using bifunctional crosslmkmg reagents Many such reagents are commercially available (e g , Pierce Chemical, Rockford, IL) and standard coupling procedures are supplied by the manufactur- ers Before screening a library, one has to make sure that the coqugation method does not impair the bmdmg property of the ligate This can usually be accom- plished by an ELISA assay using a 96-well plate coated with a known hgand (see Note 1) 2 Transfer l-10 mL of the bead-library (200,000 to 2 million beads) to a 50- lOO-mL polypropylene container Slowly dilute the dimethylformamtde (DMF) by adding an incremental amount of double-distilled water. Wash the bead- library thoroughly with double-distilled water m a column (e g., Econo column, Bio-Rad, Hercules, CA) Coat the bead-library with 0 1% gelatin (w/v) m water for at least 1 h. Wash the bead-library with PBS-Tween Transfer the library back into the polypropylene contamer with the bmdmg buffer (see Note 2) Add the ligate-alkaline phosphatase coqugate into the library with gentle mixing for 1 to 24 h at room temperature (see Note 3). 3. Transfer the bead-library to the column and wash the beads thoroughly with PBS- Tween. Then wash the bead-library one last time with TBS. Enzyme-Linked Calorimetric Library Screening 9 Fig. 1. (A) Photomicrograph of a typical enzyme-linked calorimetric bead-library screen; a positive bead is noted in the middle of the micrograph. (B) Single positive beads can easily be retrieved with a handheld micropipet under a dissecting microscope. 4. Transfer and wash the bead-library to lo-20 polystyrene Petri dishes (100 x 20 mm) with the BCIP/alkaline phosphatase buffer (see Notes 4 and 5). More dishes may be needed if the beads are too crowded and there are too many positive beads. Let the enzyme-linked color reaction develop for 30 min to 2 h. Stop the reaction by acidifying the BCIP/alkaline phosphatase buffer with several drops of 1 M HCl. Figure 1A shows the photomicrograph of a typical bead-library screen. 5. With the aid of a light box and a micropipet (e.g., Pipetman PlO, Gilson), transfer the turquoise beads into a small Petri dish. Many colorless beads will also be transferred during this process. 6. Place the small Petri dish of positive beads under a dissecting microscope and pipet individual turquoise beads to a small Petri dish of 6 M guanidine-HCI, pH 1 .O (Fig. 1B). At this stage, transfer only the positive beads (see Note 6). After 10 Lam 20-30 mm at room temperature m 6 Mguanidme-HCI, transfer the posmve beads to a dish of double-dtstrlled water. Then prpet each posrttve bead onto a glass filter and msert mto the protein sequencer cartrtdge for mtcrosequencmg (see Notes 7 and 8) 3.2. Screening with an Unlabeled Ligate by Probing with an Enzyme-Linked Secondary Antibody 1 Prepare the library as m Subheading 3.1., item 2. 2 Add the alkaline phosphatase-linked anti-ligate antibody to the bead library and incubate m bmdmg buffer for l-2 h at room temperature 3 Wash the bead-library thoroughly with PBS-Tween and finally once with TBS 4. Add BCIP substrate to the library as described m Subheading 3.1., item 4 5 After 30 mm to 2 h, stop the colortmetrrc reaction by adding several drops of 1 M HCl to each Petri dish. Remove all the color beads from the library over a light box with a mrcropipet These color beads interact with the secondary antibody alone and may be discarded. 6 Recycle the remaining colorless library with the following steps: Incubate the library with 6 M guamdine-HCl, pH 1 .O, 20-30 min, wash 5 times with double- distilled water, mix the library with DMF for 1 h, wash 5 times wrth double-distilled water, followed by PBS-Tween 7 Add the unlabeled ligate to the bead-library and incubate l-24 h at room temperature 8 Wash the bead-library thoroughly with PBS-Tween 9. Add the alkaline phosphatase-linked antrligate antibody to the bead-library and incubate l-2 h (see Note 3) Then wash the bead-library thoroughly wtth PBS- Tween and finally once with TBS. 10 Add BCIP substrate to the library as described m Subheading 3.1., item 4. After 30 min to 2 h, stop the colorimetrtc reaction by adding several drops of 1 M HCl to each Petri dish. Since the library has been prescreened with the secondary antibody alone, the posrttve beads Identified at this time should be a result of bmdmg to the ligate and not to the secondary antibody. 11. Isolate those individual posrtive beads for microsequencmg as descrrbed m Subheading 3.1., items 5,6. 4. Notes 1 For the two-step screening process, instead of using the lrgate/antr-ligate-enzyme system, one may use a biotmylated-ligate/streptavrdm-enzyme system 2. Most of the methods employed m Western blot or ELISA for lowering the back- ground can be applied to the screening of the bead-library We routmely add high salt (2X PBS), 0 1% Tween-20, and 0 1% gelatin to the bmdmg buffer Bovme serum albumin instead of gelatin has also been used successfully. 3 In order to mmimtze the background and false posmves, the concentration of ligate, ligate-enzyme conjugate, or antibody-enzyme conjugate used m the screening should be as dilute as possible Sometrmes lt is advantageous to use a [...]... with different types of peptide libraries (A) Combinatorial library XXB,B,XX, (B) mutational analysis of the TGFa epitope VVSHFND, (C) TGFa-derived peptide scanning library (7-mers, 6 amino acids overlapping starting with the upper left spot) torial peptide library XXB ,B,XX allowed the a priori delineation of the TGFa epitope The peptide scanning library consisting of overlapping peptides spanning the... the manual synthesis of a combinatorial peptide library of the type XXB,B,XX Subheading 3.7 contains details about the automated synthesis of peptide libraries using the Spot synthesizer Auto-Spot Robot APS 222 of Abimed GmbH In Subheadings 3.8 and 3.9 the synthesis of a mutational analysis and a peptide scanning library is described The screening methods of these different peptide libraries are explained... heat shock protein -peptide mteractlons (II) As another biologically relevant application, these libraries were applied for the study of metal -peptide interactions For example, we identified technetium-99m binding peptides important for tumor diagnosis (12) and nickelbinding peptides that can be used for the purification of recombinant proteins (3) The synthesis of cellulose-bound peptide libraries is... achieved on cellulose membranes (3,13,14) Described here are protocols for the manual synthesis of a combmatorral peptrde library XXB,B2XX (B = defined position, X = randomized position) (2,15,16), a peptide scanning library, and a mutational analysis of a peptrde eprtope An outline of the synthesis strategy IS given m Fig 1 Furthermore, we provide protocols for the screening of these libraries with protein... Methods 3.1 Library Synthesis For general procedures on solid phase peptide synthesis, readers are referred to (16) and (17) The peptide mixtures making up the PS-SCLs are synthesized by simultaneous multiple peptide synthesis (SMPS) (18) Mixture posrtions (X) are incorporated by couplmg mixtures of protected ammo acids for pepttdes, or aldehydes, carboxylic acids, and so forth for nonpeptides, using... BloTools GmbH, Berlin, Germany) 3 Peptlde synthesis chemicals of the previous sections 2.8 Synthesis of a Mutational Peptide synthesis 2.9 Synthesis of the previous of a Peptide Scanning Peptide synthesis 2 IO Screening chemicals Analysis chemicals sections Library of the previous sections of the Peptide Libraries 1 Methanol 2 Tns-buffered salme (TBS) 50 mA4 Tns-(hydroxymethyl)-ammomethane, 137mM NaCl, 2... acids for individual peptides 10 Synthesize all combinations of the most active mixture(s) for each of the six positrons as individual peptides (Table 3) The numbers of individual peptides to be synthesized rises exponentially with the number of amino acids chosen (1 e , one ammo acid from each posmon generates one peptide, two amino acids from each position generates 64 [26] peptides, and three ammo... libraries, such as combinatorial libraries, peptide scanning libraries, mutational analyses of peptides, loop libraries, and so forth These files can be easily loaded into the Spot-synthesis software of the Auto-Spot Robot APS 222 34 3.8 Synthesis Kramer of a Mutational and Schneider-Mergener Analysis For the manual synthesis of a mutational analysis of a linear peptide use a matrix of 21 x peptide length... minimal hgand binding protein regions is more subtly differentiated The software DIGEN generates sequence lists of peptide scanning libraries The manual synthesis of a peptide scanning library requires a much higher degree of concentration than the synthesis of a combmatorial peptide library or a mutational analysis owing to the irregular order of pipeting steps in each synthesis cycle To avoid mistakes,... F , and Lebl, M (1995) Application of a dual color detection scheme m the screening of a random combmatorial peptide library J Immunol Methods 180,219-223 3 Synthesis and Screening Combinatorial Libraries of Positional Scanning Colette T Dooley and Richard A Houghten 1 Introduction Synthetic combinatorial libraries (SCLs) are collections of very large numbers of synthetic compounds, in which all possible . One-Compound Combinatorial Peptide Library Kit S. Lam and Michal Lebl 1. Introduction The four general methods to generate and screen a huge combinatorial pep- tide library +-lo7 peptides) are:. Pentapeptide Library As indicated earlier, a solid-phase split synthesis method (4,8,14) is used to generate a random peptide library. The composition and final structure of the peptide library. 3.2. Synthesis of a Cyclic Peptide Library The synthesis of a cyclic peptide library (disulfide bond formation) is essen- tially the same as that of the linear library except that Fmoc-Cys