Peptide-Based Drug Design

Solid-Phase Synthesis of O-Phosphopeptides 213
a) Eluent A: Water (see Notes 7 and 8) containing 0.1% TFA as ion pair reagent.
b) Eluent B: 60% aqueous acetonitrile containing 0.1% TFA (see Note 9).
5. Analytical separation: Inject 20- to 50-�g sample and keep the starting conditions
of 5% eluent B for 2 min. Elute the peptides with a linear increase of 3% eluent B
per min to 95% eluent B using a flow rate of 1 mL/min. Hold these conditions for
5 min before equilibrating the column again with 5% eluent B for at least 15 min.
6. Preparative separation: Purify up to 40 mg crude peptide in a single separation
using 5% eluent B as starting conditions. After 2 min increase the content of eluent
B to 95% using a linear gradient of 1–3% acetonitrile per minute and a flow rate
of 10 mL/min (see Note 10). The slope of the gradient depends on the number of
by-products eluting close to the target peptide. Hold the final conditions of 95%
eluent B for 5 min before equilibrating the column with the starting conditions of
5% eluent B.
7. Detection: Absorption at 220 nm (see Note 11).
2.5. Mass Spectrometry
1. MALDI-TOF/TOF-MS (4700 Proteomics Analyzer, Applied Biosystems GmbH,
Darmstadt, Germany).
2. �-Cyano 4-hydroxy-cinnamic acid (CHCA) matrix:
a) Weigh 2 mg α-cyano 4-hydroxy-cinnamic acid (Bruker Daltonics GmbH,
Bremen, Germany, store at 4 ◦ C) in a microcentrifuge tube and dissolve in
250 �L water containing 0.1% TFA.
b) Vortex the solution for 10 s.
c) Add 250 �L acetonitrile (HPLC grade).
d) Vortex well until the matrix is completely dissolved.
e) Store at −20 ◦ C. This solution can be used for one month.
3. �-Cyano 4-hydroxy-cinnamic acid with phosphate (pCHCA) matrix:
a) Prepare a fresh matrix solution as described above.
b) Add ammoniumphosphate (ultra ≥99.5%, Fluka) to the matrix solution to a
final concentration of 10 mM.
c) Store at −20◦C. This solution can be used for one month.
4. 2,5-Dihydroxybenzoic acid (DHB) matrix:
a) Weigh approximately 5 mg 2,5-dihydroxybenzoic acid (Bruker Daltonics, store
at 4 ◦ C) exactly in a 1.5-mL Eppendorf tube.
b) Add 450 �L water and 450 �L methanol (calculated for 5 mg matrix, adjust
according the weighed amount).
c) Vortex well until the matrix is completely dissolved.
d) Store at −20 ◦ C. This solution can be used for up to one month.