Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 33
by comparing the MS/MS spectra of the isolated peptide and the synthetic
peptide.
2. Materials
2.1. Solid Phase Extraction
1. ZipTip TM (Millipore Corporation, Billerica, MS) containing 0.6 �L C18-phase.
2. Wetting solution: 50% aqueous acetonitrile (CH3CN, LC-MS grade; Biosolve BV,
Valkenswaard, Netherlands). Store at room temperature (see Note 1).
3. Wash solution: 0.1% aqueous formic acid (HCOOH) (puriss. p.a., for mass
spectroscopy; Fluka Chemie GmbH, Buchs, Switzerland). Store at 4 ◦ C.
4. Elution solution: 50% aqueous acetonitrile containing 0.1% formic acid.
2.2. MALDI Mass Spectrometry
1. MALDI-TOF/TOF-MS (4700 Proteomics Analyzer, Applied Biosystems Applera
Deutschland GmbH, Darmstadt, Germany).
2. MALDI matrices
3. CHCA matrix solution:
a. Weigh approximately 3.5 mg �-cyano 4-hydroxy-cinnamic acid (CHCA;
Bruker Daltonics GmbH, Bremen, Germany; store at 4◦C) in a 1.5-mL
polypropylene vial.
b. Add 250 �L of 0.1% aqueous trifluoroacetic acid (TFA) (for UV-spectroscopy,
≥99.0% (GC); Fluka Chemie GmbH) (see Note 2) or adjust this volume
according to the correct CHCA weight.
c. Vortex the solution for 10 s.
d. Add 250 �L acetonitrile (MS grade).
e. Vortex well until the matrix is completely dissolved.
f. This solution containing CHCA at a concentration of 7 mg/mL can be stored at
−20◦C for one month. This matrix solution is only for the derivatization with
2-sulfobenzoic acid cyclic anhydride.
g. For all other measurements dilute the matrix with 50% aqueous acetonitrile
containing 0.1% TFA to 4 mg/mL.
4. DHB matrix solution:
a. Weigh approximately 5 mg of 2,5-dihydroxybenzoic acid (DHB; Bruker
Daltonics GmbH; store at 4 ◦ C) in a 1.5-mL polypropylene vial.
b. Dissolve the 5 mg DHB in 450 �L water or correct the volume according to
exact weight.
c. Add 50 �L methanol (Biosolve BV, Valkenswaard, Netherlands, absolute,
HPLC grade).