Peptide-Based Drug Design

34 Stegemann and Hoffmann
d. Vortex well until DHB is completely dissolved.
e. This DHB solution (10 mg/mL) can be stored at −20 ◦ C for one month.
2.3. ESI Mass Spectrometry
1. QSTAR Pulsar I (Applied Biosystems GmbH), which is a hybrid mass
spectrometer consisting of a quadrupol and an orthogonal time-of-flight mass
analyzer (QqTOF-MS). For data processing use the Analyst QS (version 1.0)
and the Bioanalyst QS (version 1.0) software packages provided with the
instrument.
2. For static nanoESI-MS connect the static nanospray source (Protana, Odense,
Denmark) to the mass spectrometer and use for each sample to be analyzed a
new borosilicate spray capillary (ES380 “Medium”, PROXEON Biosystems AIS,
Odense, Denmark; see Note 3).
3. Spray solution: 60% aqueous acetonitrile containing 1% formic acid.
4. To couple a gradient HPLC system to the mass spectrometer, replace the needle
adapter for static nanospray by a tip adapter and connect the column to Distal
Coated SilicaTips TM with an ID of 10 �m (New Objective, Woburn, MA).
5. For highest sensitivity use a nanoHPLC system, such as an Agilent 1100 series
nanoHPLC system consisting of an isocratic pump (G1310A), an autosampler
(G1377A), two pumps (G2226A) to form the gradient controlled by ChemStation
for LC (Rev. A.10.02).
6. Sample plate: 384-well microtiter plate (Greiner Bio-One GmbH, Frickenhausen,
Germany).
7. Trap column: Zorbax 300SB-C18 (300 �m internal diameter, 5-mm length,
particle size 5 �m; Agilent Technologies GmbH).
8. Eluent A: 3% aqueous acetonitrile containing 0.1% formic acid.
9. Separation column: Zorbax 300SB-C18 (internal diameter of 100 �m, column
length 150 mm, particle size 3.5 �m; Agilent Technologies GmbH).
10. Eluent B: Acetonitrile containing 0.1% formic acid.
2.4. Reduction and Alkylation of Disulfide Bonds
1. ABC buffer: 50 mM aqueous ammonium bicarbonate (NH4HCO3) (BioChemika
Ultra, ≥99.5%, Fluka Chemie GmbH; store at room temperature). Check that pH
is between 7.2 and 7.8. Store at room temperature.
2. DTT solution: Dissolve 15.42 mg 1,4-dithiothreitol (≥99% p.a., Carl Roth GmbH
& Co. KG, Karlsruhe, Germany; store at 4◦C) in 100 �L water. Prepare fresh for
each reaction. Do not store.
3. Acrylamide solution: Serva Acrylamide 4X solution (Serva Electrophoresis
GmbH, Heidelberg, Germany) corresponding to a 40% (w/v) acrylamide solution.
Store at 4◦C.