Peptide-Based Drug Design

36 Stegemann and Hoffmann
2. SACA reagent: 2-Sulfobenzoic acid cyclic anhydride (SACA) (technical, ≥95.0%,
Fluka Chemie GmbH; store at room temperature) at a concentration of 2 mg/mL
in dry tetrahydrofurane (THF, extra dry with molecular sieve, water < 50 ppm,
stabilized; Acros Organics, Geel, Belgium). Prepare always a fresh solution and
use immediately.
3. Methods
3.1. Solid Phase Extraction on a Reversed Phase
1. Add 5 �L elution solution to the bottom of a 0.6-mL polypropylene vial (see
Note 6) and close the vial. Use 10-�L pipette tips for this and all following steps.
2. Dissolve the peptide in 10 �L wash solution.
3. Wash the ZipTipTM first by aspirating and dispensing 10 �L wetting solution to the
waste. Repeat this washing procedure at least twice (see Note 7).
4. Equilibrate the stationary phase by drawing in wash solution and dispensing it to
the waste three times.
5. Put the tip of the ZipTipTM into the peptide solution to be purified and aspirate
and dispense the solution slowly at least ten times. Thus the peptide is completely
bound to the stationary phase.
6. Remove all polar impurities and salts from the stationary phase by washing the
packing material with 10-�L aliquots of wash solution. Repeat this procedure
seven times.
7. Slowly aspirate the 5 �L of elution solution stored in the 0.6-mL polypropylene
vial (step 1) and dispense it again in the tube. Repeat at least five times without
getting any air into the packing material.
8. Aspirate 5 �L of fresh elution solution and dispense once into the 0.6-mL
polypropylene vial yielding 10 �L peptide solution to be analyzed either by
MALDI-MS or static nanoESI-MS (see Note 8).
3.2. MALDI Mass Spectrometry
3.2.1. Sample Preparation with CHCA as Matrix
1. Spot 0.5 �L of the CHCA matrix solution on the MALDI target (see Note 6).
2. Wait until the spot is dried (see Note 9)andadd1�L of the sample solution in the
middle of the crystallized matrix spot (see Note 10).
3. Wait until the spot is dried again (about 5 min) (see Note 9). Check with a microscope
that light yellow crystals were formed (see Note 11).
3.2.2. Sample Preparation with DHB as Matrix
1. Spot 0.5 �L of the DHB matrix solution on the MALDI target.
2. Add 1 �L of the sample solution in the middle of the dried matrix spot (see
Note 10).
3. Wait until the spot is dried (see Note 9).