Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 45
14. To achieve a faster separation it is also possible to start the gradient earlier to
compensate the dead volume between the gradient mixer and the trap column.
On our instrument we have a dead volume of about 1.3 �L. At a flow rate of 300
nL/min we start the gradient 4 min before the valve switches.
15. Due to the high sensitivity it is favorably to couple a nanoHPLC to an ESI-source.
As mass spectrometers are concentration dependent detectors, the sensitivity of
an instrumental setup is mostly determined by the peptide concentration of the
eluate but not by the peptide amount. Thus a nanocolumn with a flow rate of
300 nL/min provides an about thousand times higher sensitivity than a microbore
column with a flow rate of 300 �L/min. As an alternative to buying a nanoHPLC
system it is also possible to use a relatively inexpensive flow splitter after the
pump and before the injection valve and the column. Thereby the flow rate can
be reduced to use a capillary column (flow rate 4 �L/min) on an analytical HPLC
system or a nanocolumn on a capillary HPLC system. Instead of a flow-splitter
it is preferred to couple a nanoHPLC to an ESI-source. Thereby, the flow rate
is split according to the column backpressure, i.e., mostly the column volumes
if the same packing materials are used. However, these low-cost setups are less
reliable than a nanoHPLC and the reproducibility is worse.
16. Alternative to DDA, the term “information-dependent acquisition” (IDA) is used.
Both DDA and IDA describe an automatic mode where the MS/MS parameters
are collected automatically by calculating the parameter set (e.g., collision
energy) based on the mass and charge state of the selected precursor ion.
17. The described conditions alkylate cysteine residues almost quantitatively, even
for defensins, which contain three disulfide bonds. Higher temperatures (>37 ◦ C)
and longer reaction times (> 1 h) do not further increase the cysteine
alkylation but may produce further byproducts, such as carboxymethyllysine
(CML).
18. Despite the high lysine and arginine content of most antimicrobial peptides,
tryptic digests typically yield medium-sized peptides, as often a proline residue
follows these basic residues eliminating the cleavage site.
19. Prepare a fresh OME solution for every reaction. Storage of this solution reduces
the guanidination degree of the lysine residues significantly yielding heterogeneous
samples. To process several samples in parallel prepare a larger volume of
the solvent (i.e., 30 �L water, 20 �L 0.1% TFA, and 55 �L ammonium hydroxide
solution) and dissolve the O-methylisourea hemisulfate (2.55 mg/51 mL in Tris-
HCl buffer) just before the guanidation reaction is started, add 15 �L tothe
solvent solution. Add 12 �L to each dried peptide.
20. Do not press the tip of a ZipTip TM on the bottom of a polypropylene vial or any
other surface, as the packing material might break and block the tip. Also remove
it carefully with a pair of tweezers and mount it on the pipette by touching the
ZipTip TM only on the top.
21. Even if a modified peptide was only detected in negative ion mode it is still
possible to record a tandem mass spectrum with reasonable signal intensities in