Peptide-Based Drug Design

32 Stegemann and Hoffmann
promising candidates are gene-encoded antimicrobial peptides isolated from a
wide variety of different species, such as insects, birds, reptiles, amphibics,
fishes, and mammals (4,5). These naturally occurring AMPs form a first line
of host defense against pathogens and are involved in innate immunity. They
are divided into several subgroups based on their amino acid composition and
structure (6). Among the several thousand sequences possessing antimicrobial
activities, only a few peptides have been thoroughly investigated using a broad
spectrum of bacteria or fungi. Whereas all these peptides appear to attach
first to the membrane, they kill the bacteria by different mechanisms, such
as forming pores in the membrane or targeting specific intracellular bacterial
targets.
The search for new AMPs starts from body fluids, cells, organs, or tissues,
for example, to isolate active peptides and small proteins to homogeneity
combining different orthogonal separation techniques, such as liquid chromatography
(size exclusion, ion-exchange, reversed-phase chromatography) or gel
electrophoresis (SDS-PAGE). Fractions or bands showing antimicrobial activities
against a selected panel of microbes are further separated until a single
active peptide is finally obtained, typically at the low �g orevenngscale,
judged by matrix-assisted laser desorption/ionization (MALDI) or electrospray
ionization (ESI) mass spectrometry (MS). Besides the mass information
of the intact peptide, these techniques can also be used to determine their
sequences using tandem mass spectrometry. These instruments rely on various
combinations of mass analyzers, such as ion traps (IT), triple quadrupole
(QqQ), hybrid quadrupole/time-of-flight (QqTOF), or time-of-flight/time-offlight
(TOF/TOF) instruments. Hybrid instruments provide especially good
access to the peptide sequences due to their high sensitivity and mass
accuracy.
The product ions produced from the selected precursor ion contain all information
to deduce the peptide sequence from the b- or y-ion series (7,8) resulting
from cleavage of the peptide bonds by collision-induced dissociation (CID), also
called collision-activated dissociation (CAD). In principle, the peptide can be
identified from a single MS/MS spectrum automatically if the corresponding
protein is found in a data base (9). If not, the complete sequence has to be
retrieved from the mass spectrum de novo. However, this requires quite a lot of
experience and is typically limited to peptide lengths from approximately 10 to
25 residues. Both longer and shorter peptides are difficult to sequence. Thus, it
is often obligatory to digest the sample and sequence the shorter peptides, which
are combined afterwards to the complete sequence. Moreover, spectral interpretations
are often ambiguous due to overlapping ion series, isobaric amino acids,
or a low mass accuracy. Therefore, we always confirm the postulated sequence