Peptide calibration standard mixture

The substrate specificity of CPZ was evaluated against different synthetic peptides. For this purpose, two synthetic Met-enkephalin peptides differing only in the C-terminal residue (YGGFMKR and YGGFMKK) and a Met-Enkephalin with two C-terminal Arg (YGGFMRR) were purchased from Phoenix pharmaceuticals (Phoenix Pharmaceuticals Inc., Mannheim, Baden-Württemberg, Germany). In addition, a fourth synthetic peptide with a C-terminal Glu (ARLSQKFPKAE) was acquired from GenScript (GenScript Biotech, Piscataway, NJ, USA). Approximately 1.6 μM of each peptide was incubated with 100 nM of CPZ in 100 mM Tris-HCl, pH 7.5, 150 mM NaCl buffer at 37 °C up to 24 h. Aliquots of 2 µL were taken after different incubation times (0, 30, 60, 120, and 300 min), immediately stopped by addition of four volumes of 0.1% TFA. Samples from each reaction were mixed with an equal volume of α-cyano-4-hydroxycinnamic acid (hcca) matrix solution, spotted onto a 384 target plate polished steel (Bruker Daltonics, Billerica, MA, USA), and evaporated to dryness at room temperature. Mass spectra were recorded on an ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in reflectron positive ion mode, and at 25 kV. A standard peptide calibration mixture (Bruker Daltonics, Billerica, MA, USA) was used as a reference.

Protein spots that showed a significant difference (P < 0.05) in the 2-D electrophoresis experiment were excised manually, and in-gel digested overnight at 37 °C with sequencing grade modified trypsin (Promega, USA). Tryptic peptides (0.5 mL) were mixed with a saturated solution of α-Cyano-4-hydroxycinnamic acid (CHCA) in 50% (w/v) acetonitrile containing 0.1% trifluoroacetic acid. The mixture was spotted onto a MALDI sample plate and crystallized at room temperature. The same procedure was used for the standard peptide calibration mixture (Bruker Daltonics, Germany). Mass spectra were acquired using a MALDI-TOF-MS Autoflex spectrometer (Bruker Daltonics, Germany) operating in the PMF fully automated mode, or manually in the LIFT mode in the case of MALDI-TOF/TOF. PMFs and MS/MS ions were searched against the NCBI nr/Proteobacteria database using MASCOT software (Matrix Science, UK). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) covering a total of 2270 B. japonicum genes and showing a complete or nearly complete pathway in several cases43 (link) was used to identify the possible pathways affected by WSHM treatment. Enzymes and proteins annotated under the global KEGG category metabolism were identified.

The samples from each of the ten iTRAQ experiments were subjected to nano-LC
separation using an EASY-nLC Proxeon (Bruker Daltonics, Germany) coupled to a
Proteineer fc II (Bruker Daltonics, Germany) fraction collector as previously
described34 (link). For each iTRAQ experiment,
5 μl of respectively KCl fraction was
injected in triplicate. In total, 384 fractions were collected. The
UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics, Germany) instrument was operated
in the positive ion mode and controlled by the Compass for Flex software,
version 1.3 (FlexControl 3.3, FlexAnalysis 3.3, Bruker Daltonics, Germany). Five
thousand laser shots were accumulated per spectrum in the MS and MS/MS modes.
The spectrometric analysis was performed in automatic data-dependent mode. The
nonredundant precursor peptides were selected for MS/MS analysis using the
WARP-LC 1.3 software (Bruker Daltonics) with a signal-to-noise threshold of 12.
The MS spectra were externally calibrated using the Peptide Calibration Standard
mixture (Bruker Daltonics).