Peptide-Based Drug Design

Bioactive Peptides in Invertebrate Immune Systems 25
120 experimentally infected flies) was loaded onto a microbore RP column
(Aquapore RP 300 C8, 1× 100 mm, Brownlee). Elution was performed either
with a linear gradient of 0–80% acetonitrile in acidified water over 80 min (19,27)
or with a linear gradient of 2–62% acetonitrile in acidified water over 60 min
(30), both at a flow rate of 80 �L/min at 35 ◦ C.
11. If the molecule of interest is eluting during the first HPLC run with the front, the
following purification step should be performed in an isocratic condition with a
minimum of acetonitrile (0.5%) or directly in acidified water without acetonitrile.
12. Using RP-HPLC, the alkylated peptide is separated from reagents and the
unreacted peptide with a gradient of acidified acetonitrile from 2 to 80% in
120 min. The large quantity of reagents is eliminated owing to long washing
period (at least 30 min) with 2% acidified acetonitrile prior starting the gradient.
Using the 4-vinylpyridine, an increase in the absorbance is observed on the
alkylated peptide compared to the native molecule. When using magnetic beads,
the chemically modified peptide is recovered by elution with 60% acetonitrile.
13. For molecular mass fingerprint of an enzymatic digestion, the acidity of the
matrix and of the 1% TFA droplet is sufficient to quench any further digestion.
14. For determining the molecular mass of a polypeptide, a single-stage mass
spectrometer is appropriate, whereas for analysis of structural features tandem
MS (MS n ) is required. In this latter case, after molecular mass measurement,
specific ions are selected and then subjected to fragmentation through collision in
a specific chamber (collision cell) supplied with an appropriate gas (e.g., argon).
Instrument performance (resolution, sensitivity, mass accuracy) depends on the
instrument type, the ionization method, and the scanning capabilities.
15. For the analysis of a trypsin digest by MS, an internal calibration is performed
with fragments of autolysed trypsin (e.g., [M-H] + at m/z 842.510, 1045.546, and
2211.104).
16. To avoid the inconvenience of the TFA (decrease sensitivity) during MS, if
molecular mass measurements are performed on fractions that may contain traces
of TFA, the sample has to be desalted and concentrated onto a C18 Zip-Tip.
Elution of the peptide(s) will then be performed by 50% aqueous acetonitrile
supplemented with 1% formic acid.
5. Conclusion
Intensive research efforts for developing new anti-infectious and antitumoral
drugs for human health rely on technological advancements in screening of
combinatorial chemical libraries and/or natural extracts generated from tissues
and fluids from animals and plants. Presently, the use of natural extracts and
the random HTS of libraries of synthetic or natural molecules have successively
allowed the discovery of new therapeutic molecules. The race to discover
the new drugs of tomorrow has begun. In the area of antibiotics, facing the
increased resistance of bacteria and fungi to conventional antibiotics, the natural