Autoflex 3 smartbeam spectrometer

The fractions obtained from the L. labyrinthicus skin secretion by chromatographic separation were analyzed by mass spectrometry performed on a MALDI-ToF/ToF mass spectrometer (Autoflex™ III SmartBeam spectrometer, Bruker Daltonics, Germany) in linear and reflector modes and the spectra were processed with MassLynx^TM3.5 (UK) and FlexAnalysis 3.3 (Bruker Daltonics, Germany).
Briefly, solubilized fractions (0.5 μL of sample, variable concentrations) were spotted onto the target followed by 0.5 μL of CHCA (α-cyano-4-hidroxycinnamic acid) or DHB (2,5-dihydroxybenzoic acid) matrix solution (60% acetonitrile/0.3% TFA), and allowed to dry at room temperature (dried-droplet method). Peptide Calibration Standard II (700–4000 Da) and Protein Calibration Standard I (3000–25,000 Da) (Bruker Daltonics, Germany) were used as external calibration standards. Mass spectra from the average of 256 laser pulses from m/z 600 to 39,400 were obtained.

The peptides, with amidated C-terminus, were prepared by solid-phase synthesis on a Rink amide resin by using the Fmoc strategy [27 ]. Couplings were performed with N,N’-diisopropylcarbodiimide/1-hydroxybenzotriazole in N,N-dimethylformamide for 120 min under stirring (240 rpm). Cleavage and final deprotection were conducted with TFA:triisopropylsilane:ethanedithiol:water (94.0:1.0:2.5:2.5, v:v:v:v) for 180 min at room temperature. The peptide products were precipitated with diisopropyl ether, extracted with water and lyophilized. Then, peptides were purified by RP-HPLC (Varian Pro Star 210 Series, USA) using a preparative C18 column (Vydac C18, 300 × 7.8 mm, USA) eluted by a linear gradient of acetonitrile containing TFA 0.1% (solvent B) (0–5 min, a gradient of 20–35% acetonitrile in 0.1% TFA in water; 5–20 min, gradient of 35–45% acetonitrile containing 0.1% TFA in water; 20–35 min, 45-100% acetonitrile containing 0.1% TFA in water; 35–37 min, 100% acetonitrile with 0.1% TFA; 37–40 min, 100–20% acetonitrile containing 0.1% TFA in water). A flow of 2.0 mL.min⁻¹ was used and the peptides were detected at 214 nm. The identities of the peptides were confirmed by MALDI-ToF/ToF mass spectrometry (autoflex™ III SmartBeam spectrometer, Bruker Daltonics, Germany).

In vitro phosphorylation assays were carried out with 100 μM unlabeled or ¹³C,¹⁵N-labeled H1H2 in 50 or 200 μL phosphorylation buffer (25 mM 3-(N-morpholino)propanesulfonic acid, 25 mM MgCl₂, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 2 mM EDTA, 10 mM adenosine triphosphate, 5 mM DTT, pH = 7.2), respectively. To this, 0.3 µg of active, GST-tagged, human Akt1 (Sigma-Aldrich) or 0.3 µg active, GST-tagged, human PKC δ (Biaffin GmbH) was added and incubated at 37°C for up to 120 hr. The unlabeled samples were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry on a Bruker autoflex III smartbeam spectrometer with α-cyano-4-hydroxycinnamic acid as matrix after 24 hr. The ¹³C,¹⁵N-labeled samples were analyzed by NMR spectroscopy after 120 hr and partially assigned as described above.