Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 35
4. Iodoacetamide solution: Dissolve 27.5 mg iodoacetamide (purum ≥98.0%, Fluka
Chemie GmbH; store at 4 ◦ C) in 100 �L water. Store at −20 ◦ C(see Note 4).
2.5. Enzymatic Digests
1. Three millimolar ABC-buffer: 3 mM aqueous ammonium bicarbonate buffer
(BioChemika Ultra, ≥99.5%, Fluka Chemie GmbH). Check that pH is between
7.2 and 7.8. Store at room temperature.
2. Trypsin solution: Dissolve trypsin (Promega Sequencing Grade Modified Trypsin
Porcine, activity: 5000 U/mg; store at −20 ◦ C) in the 3 mM ABC-buffer to obtain
a final concentration of 20 ng/�L. Store in aliquots at −20 ◦ C(see Note 5).
3. Ten millimolar ABC-buffer: 10 mM aqueous ammonium bicarbonate buffer
(BioChemika Ultra, ≥99.5%, Fluka Chemie GmbH). Check that pH is between
7.2 and 7.8. Store at room temperature.
4. Lys-C solution: Dissolve endoprotease Lys-C from Lysobacter enzymogenes
(sequencing grade, Roche Diagnostics GmbH, Roche Applied Science,
Mannheim, Germany; store at −20 ◦ C) in the 10 mM ABC-buffer to a final concentration
of 10 ng/�L. Store in aliquots at −20 ◦ C(see Note 5).
2.6. Derivatization of Amino Groups
2.6.1. Guanidation of Lysine (10)
1. OME-reagent: Dissolve 50 mg O-methylisourea hemisulfate (94.0%, Acros
Organics, Geel, Belgium; store at room temperature) in 51 �L water. Prepare
always a fresh solution and use immediately.
2. Seven molar aqueous ammonium hydroxide solution. Store at room temperature.
3. 0.1% aqueous TFA.
2.6.2. N-Terminal Peptide Derivatization Using 4-Sulfophenyl
Isothiocyanate (11)
1. Tris-buffer: 50 mM Tris-HCl buffer (pH 8.2). Store at room temperature.
2. SPITC-reagent: Dissolve 2.55 mg 4-sulfophenyl isothiocyanate sodium salt
monohydrate (SPITC, technical grade, Sigma Aldrich, St. Louis, MO; store at
4 ◦ C) in Tris-buffer at a concentration of 2.55 mg/mL. Prepare always a fresh
solution and use immediately.
3. Eighty percent aqueous acetonitrile containing 0.5% trifluoroacetic acid. Store at
4 ◦ C.
2.6.3. N-Terminal Peptide Derivatization Using 2-Sulfobenzoic Acid
Cyclic Anhydride (12)
1. Reaction buffer: Mixture of acetonitrile and 12.5 mM aqueous ammonium bicarbonate
buffer (1:1 by vol.) (BioChemika Ultra, ≥99.5%, Fluka Chemie GmbH;
store at 4◦C).