Peptide-Based Drug Design

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14 Bulet 2.5.2.1. MALDI-TOF-MS 1. MALDI-TOF-MS may be operated in either linear or reflector mode. Due to the cationic character of the peptides investigated, detection is performed in a positive mode. 2. Appropriate standard peptides for calibration within the mass range 500–5000 in reflector mode and 2000 to 20–30 kDa for the linear mode. 3. A matrix adapted for
Bioactive <strong>Peptide</strong>s in Invertebrate Immune Systems 15 e. Staphylococcus aureus V8 proteinase (50 mM ammonium carbonate, pH 7.8, 0.01% Tween 20) that is specific for Glu-X cleavage. f. Asparaginyl endopeptidase (20 mM sodium acetate buffer, pH 5, 0.01%, Tween 20, 1 mM dithiothreitol (DTT), 1mM EDTA) that is specific for Asn-X cleavage. g. Endoproteinase Asp-N (20 mM sodium phosphate buffer, pH 8.0, 0.01% Tween 20) that is specific for X-Asp cleavage. h. Pyroglutamate aminopeptidase (100 mM sodium phosphate buffer, pH 8, 10 mM EDTA, 5% glycerol, 5 mM DTT) for removal of pyroglutamic acid ( Nterminal cyclic residue that prevents Edman degradation). i. Carboxypeptidase Y and P (50 mM sodium citrate buffer, pH 6 [for Y] and pH 4 [for P]) for C-terminal amino acid removal. j. Thermolysin (100 mM MES [2-(N-morpholine) ethane sulfonic acid, pH 6.5, 2mM CaCl2]) for disulfide bridge assignment or for generating a large series of peptidic fragments. 2.5.4. Chemical Treatment: Reduction and Alkylation To facilitate sequencing analysis and enzymatic cleavage, reduction and alkylation of the peptides to open their potential disulfide bridges is recommended. Several alkyl groups can be used: acrylamide, iodoacetamide, and 4-vinylpyridine (4-VP). S-pyridylethylation is preferred over the other alkyl groups when Edman degradation (PTH-4-pyridylethylated-Cys is commercially available) and MS (addition of 57 Da per alkylated cysteine residue) have to be performed. 1. Reaction buffer: 0.5 M Tris-HCl, 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5 containing 6 M guanidinium hydrochloride. 2. 2.2 M DTT; tris(2-carboxyethyl)phosphine hydrochloride (TCEP) may be an alternative. 3. Distilled 4-VP (see Note 1). Keep the solution under nitrogen atmosphere in a deep freezer. Iodoacetamide is also frequently used. An addition of 107 or 57 Da per alkylated cysteine residue is observed with vinylpyridine and iodoacetamide, respectively. Cysteine residues derivatized with N-methyl iodoacetamide can be also analyzed by the Edman sequencing (no PTH standard commercially available). 4. Stop solution: acidic solution, 1% TFA or 1% acetic acid or less than 3% formic acid. 5. Desalting by reversed-phase HPLC (see Subheading 2.4.) orSECormicro- SPE onto C18 Zip-Tip (used the conditions recommended by the manufacturer Millipore). The recently developed magnetic beads from Bruker may represent a fast and economical alternative. 6. Nitrogen or argon gas tank.
Page 2 and 3: Peptide-Based Drug Design
Page 4 and 5: METHODS IN MOLECULAR BIOLOGY TM Pep
Page 6 and 7: Preface Natural products chemistry
Page 8 and 9: Contents Preface...................
Page 10 and 11: Contributors Nikolinka Antcheva •
Page 12 and 13: Contributors xi Alessandro Tossi
Page 14 and 15: 2 Otvos advance of computer power a
Page 16 and 17: 4 Otvos see derivatives active in b
Page 18 and 19: 6 Otvos was designed based on ligan
Page 20 and 21: 8 Otvos 27. Borghouts, C., Kunz, C.
Page 22 and 23: 10 Bulet Key Words: Invertebrate im
Page 24 and 25: 12 Bulet 6. 5 �L or higher volume
Page 28 and 29: 16 Bulet 7. Small-volume low-protei
Page 30 and 31: 18 Bulet 3. Centrifuge between 8000
Page 32 and 33: 20 Bulet internal diameter). Increa
Page 34 and 35: 22 Bulet interest. As no instrument
Page 36 and 37: 24 Bulet 3. Incubate the plates in
Page 38 and 39: 26 Bulet bioactive peptides from th
Page 40 and 41: 28 Bulet 21. Chernysh, S., Kim, S.I
Page 42 and 43: 3 Sequence Analysis of Antimicrobia
Page 44 and 45: Sequence Analysis of Antimicrobial
Page 58 and 59: 4 The Spot Technique: Synthesis and
Page 60 and 61: The Spot Technique 49 3. For amine
Page 62 and 63: The Spot Technique 51 2. Biotinylat
Page 64 and 65: The Spot Technique 53 the solutions
Page 66 and 67: The Spot Technique 55 solution. Ens
Page 68 and 69: The Spot Technique 57 (see Fig. 3)
Page 70 and 71: The Spot Technique 59 Fig. 5. Princ
Page 72 and 73: The Spot Technique 61 library, the
Page 74 and 75: The Spot Technique 63 7. Regenerati
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The Spot Technique 65 following rul
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The Spot Technique 67 20. Atherton,
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The Spot Technique 69 50. Bolger, G
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5 Analysis of A� Interactions Usi
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Analysis of Aβ Interactions 73 are
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Analysis of Aβ Interactions 75 Ana
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Analysis of Aβ Interactions 77 3.
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6 NMR in Peptide Drug Development J
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NMR of Peptides 89 usually require
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NMR of Peptides 91 limiting the siz
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NMR of Peptides 93 and STD NMR expe
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NMR of Peptides 95 The basis of the
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NMR of Peptides 97 Fig. 5. Overlay
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NMR of Peptides 99 Fig. 7. Schemati
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NMR of Peptides 101 4.4.2. Saturati
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NMR of Peptides 103 4.6. Diffusion
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NMR of Peptides 105 Fig. 13. Propos
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NMR of Peptides 107 protein prevent
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NMR of Peptides 109 6. Wuthrich, K.
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NMR of Peptides 111 45. Morris, K.
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NMR of Peptides 113 78. Aumailley,
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116 Copps et al. Fig. 1. Potential
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118 Copps et al. Fig. 2. Flowchart
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120 Copps et al. 10. In accordance
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122 Copps et al. Fig. 3. DSSP for g
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124 Copps et al. ingly with the sam
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126 Copps et al. 19. Essman, U., Pe
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128 Hilpert et al. Key Words: Scree
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130 Hilpert et al. Table 1 (Continu
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132 Hilpert et al. different natura
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134 Hilpert et al. wt A C D E F …
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136 Hilpert et al. methods primaril
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144 Hilpert et al. Table 3 Summary
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148 Hilpert et al. of substituting
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150 Hilpert et al. in models that c
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152 Hilpert et al. than control. Gi
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154 Hilpert et al. Table 4 Accuracy
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156 Hilpert et al. 25. Pini, A., Gi
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158 Hilpert et al. 55. Giacometti,
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9 Investigating the Mode of Action
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Proline-Rich Antimicrobial Peptides
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10 Serum Stability of Peptides Håv
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Serum Stability of Peptides 179 2.
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Serum Stability of Peptides 183 �
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11 Preparation of Glycosylated Amin
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Glycosylated Amino Acid Synthesis 1
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12 Synthesis of O-Phosphopeptides o
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Solid-Phase Synthesis of O-Phosphop
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13 Peptidomimetics: Fmoc Solid-Phas
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Peptidomimetics 225 O O R S N H Pep
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Peptidomimetics 227 not without its
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Peptidomimetics 229 Oxidation step:
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Peptidomimetics 231 of peptidosulfo
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Peptidomimetics 233 8. Allow the re
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Peptidomimetics 235 3.3.2. Solid-Ph
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Peptidomimetics 237 On the other ha
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Peptidomimetics 239 nitrogen (73).
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Peptidomimetics 241 3. Cover the re
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Peptidomimetics 243 efficient synth
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Peptidomimetics 245 55. Alsina, J.,
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14 Synthesis of Toll-Like Receptor-
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Lipopeptides 249 2. Materials 2.1.
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Lipopeptides 251 Fig. 1. Structural
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Lipopeptides 253 8. To enable lipid
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Lipopeptides 255 Representative res
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Lipopeptides 257 Fig. 2. Immunogeni
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Lipopeptides 259 8. A large plastic
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Lipopeptides 261 26. Nardin, E.H.,
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264 Otvos response, synthetic pepti
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266 Otvos Fig. 3. Schematic present
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268 Otvos 3. Methods The main goal
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270 Otvos 3.2.3. Purification and Q
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272 Otvos 6. Meyer, D., and Torres,
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16 Cysteine-Containing Fusion Tag f
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Targeted Therapeutic and Imaging Ag
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Index A A� peptides aggregation a
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Index 297 phosphopeptides influenci
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Index 299 for genetically synthetic
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Index 301 peptidosulfonamide, disad
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Index 303 solid phase, 180, 214 sul
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Peptide-Based Drug Design

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