Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 37
4. Add again 0.5 �L of the DHB matrix solution. Check with a microscope that white
needle-like crystals were formed as an outer circle of the spot (see Note 11).
3.2.3. Recording MALDI Spectra
1. Calibrate the mass spectrometer with a peptide mixture spanning a similar
mass range to be used afterwards for the analysis, such as a mixture of des-
Arg bradykinin (M = 903.4603 g/mol), angiotensin I (M = 1295.6775 g/mol),
Glu-fibrino-peptide B (M = 1569.6696 g/mol), ACTH 1–17 (M = 2092.0789
g/mol), ACTH 18–39 (M = 2464.1910 g/mol), and oxidized insulin B chain
(M = 3493.6435 g/mol) (4700 Proteomics Analyzer Calibration Mixture, Applied
Biosystems GmbH; store at −20 ◦ C).
2. As the layer technique with CHCA produces a homogeneous peptide distribution
within the matrix, record the spectra from all parts of the sample spot. For samples
prepared with DHB, use only the outer edge of the matrix spot, as the center part
produces mostly salt clusters and sodiated peptide ions.
Record the spectra in reflectron mode using an acceleration voltage of 20 kV,
70% grid voltage and a delay of 1.277 ns. Generate MS spectra by accumulating
2000 laser shots (Fig. 1). Analyze the mass spectra with the Data Explorer R○
Software Version 4.6 (Applied Biosystems GmbH) (see Note 12).
Fig. 1. MALDI-TOF mass spectrum of an antibacterial peptide isolated from dog
Canis familiaris using the CHCA matrix. The dominant signals at m/z 4190.38 and
2095.68 correspond to the [M+H] + and [M+2H] 2+ ions of the same peptide. The doubly
charged ion, which is usually not obtained for peptides in MALDI-MS, is probably
detected due to the high content of basic lysine and arginine residues.