Peptide-Based Drug Design

Sequence Analysis of Antimicrobial Peptides 39
O
peptide
H
N peptide
peptide
N
H
O
S
S
peptide
DTT
O
2 peptide
H
N peptide
SH
I
O
O
NH 2
NH 2
O
peptide
H
N peptide
S
O
NH 2
O
peptide
H
N peptide
Scheme 1. Reduction of homodimer peptide linked via a disulfide bridge with dithiothreitol
(DTT) and alkylation of the resulting thiol groups of both cysteines with either
acrylamide (upper reaction) or iodoacetamide (lower reaction).
3.5. Counting Cysteine Residues
1. Determine the molecular masses of the alkylated peptide samples by nanoESI-MS
in static mode using 2-�L sample or by MALDI-MS using either CHCA matrix
or DHB matrix (see Subheadings 3.2.1. and 3.2.2.).
2. Calculate the differences of the original peptide mass and the recorded masses after
alkylation with iodoacetamide and acrylamide (e.g., the peptide mass was shifted
from 4540.35 to 4972.84 g/mol and 4888.42 after reduction and alkylation with
acrylamide and iodoacetamide, respectively, corresponding to mass differences of
432.49 and 348.07 g/mol.).
3. Determine the number of cysteine residues by dividing the mass difference calculated
for the acrylamide sample by 72.08 and 58.05 g/mol for the iodoacetamide
sample (e.g., the mass shift of 432.49 g/mol for acylamide and 348.07 g/mol
for iodoacetamide correspond to six cysteine residues in both cases, i.e., three
disulfide bonds in the original sample).
3.6. Tryptic Digest
1. If the peptide to be digested contains cysteine residues, reduce and alkylate them
prior to any enzymatic digest (see Subheading 3.4.) using only 1 �L of either of
the two alkylation reagents for 40 pmol peptide.
2. Dry the peptide solution (40 pmol) in the SpeedVac and add 8 �L 3mMABC
buffer.
3. Add 2 �L trypsin solution and mix carefully by aspirating and dispensing 2 �Lof
the solution several times with the tip.
4. Split the sample in two parts of equal volume to 0.6-mL polypropylene vials (see
Note 6).
+
+
– HI
S
O
NH 2