Hct ultra ion trap

The HPLC system (1100/1200 series, Agilent, Waldbronn, Germany) includes a binary pump (G1312A), an autosampler (G1329B), and a DAD-detector (G1316A). It was coupled to an HCT Ultra Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization source (ESI). The anthocyanins and copigments were separated on a Luna C18(2) 3 µ column (150 × 2.0 mm, Phenomenex (Torrance, CA, USA)) using water/acetonitrile/formic acid (95/3/2; v/v/v) (eluent A) and water/acetonitrile/formic acid (48/50/2; v/v/v) (eluent B) at a flow rate of 0.2 mL/min. Gradient elution was performed: 0 min 6% B, 30 min 35% B, 35 min 40% B, 45 min 90% B, 50 min 90% B, 55 min 30% B, 70 min 6% B. The ESI source was operated in alternating mode (+/−3000 V) (positive mode for anthocyanins and negative mode for copigments), using nitrogen as the nebulizer (50 psi) and drying gas (10 L/min, 365 °C). The sample extracts (around 2 mg) were dissolved in 2 mL of eluent A with a pH of 2.52 so that the flavylium cation form of the anthocyanins was stabilized. Aliquots of 5 µL of each sample were analyzed by the HPLC-PDA-ESI-MS/MS method described above according to Ostberg-Potthoff et al. [33 (link)] using the Bruker Hystar 3.2, Bruker ESICompass 1.3 for HCT/Esquire, and Data Analysis Version 3.0 software packages (Bruker Daltonics, Bremen, Germany).

The tryptic peptides were resuspended in 0.1% formic acid following the previous preparation. Triplicate samples were injected into an ion trap mass spectrometer (HCT Ultra Ion Trap, Bruker Daltonics, Germany) linked to a nano-LC system (Ultimate 3000 LC System, Thermo Fisher Scientific, Waltham, MA, USA). The peptide mixture was fractionated using a reverse-phase high-performance liquid chromatography column (Acclaim PepMap^TM 100 Å, 75 µm × 5 cm, Thermo Fisher Scientific, Leicestershire, UK and PepSwift Monolithic Trap Column 200 µm × 5 cm Thermo Fisher Scientific, Leicestershire, UK). The mobile phases consisted of buffer A (0.1% formic acid in H₂O) and buffer B (0.1% formic acid in 80% acetonitrile) as eluting peptides. The elution with linear gradient was run as follows: 4–70% of solvent B at 0–20 min (the time point of retention time), followed by 90% of solvent B at 20–25 min to remove all peptides in the column and 96% solvent A for 15 min for column re-equilibration. Finally, mass spectra of gradient eluted peptides were examined using a MS1 precursor scan (m/z 400–1500) in Data-Dependent Acquisition (DDA) mode. The five most abundant multiple charged precursor ions were selected for MS2 fragmentation from a MS2 scan (m/z 200–2800).

For mass spectrometric analyses, we used approx. 600 eggs per replicate in each group. F. hepatica eggs were lysed in SDT buffer (4% SDS, 0.1 M DTT, 0.1 M Tris/HCl, pH 7.6) in a thermomixer (Eppendorf ThermoMixer C, 30 min, 95 °C, 750 rpm) with added glass beads. After that, the samples were centrifuged (15 min, 20,000×g) and collected supernatants used for filter-aided sample preparation (FASP) as described elsewhere^{35 (link)} using 0.5 μg of trypsin per sample (sequencing grade, Promega Corporation). The resulting peptides were used for LC–MS/MS analyses. Once sample preparation was complete, the total amount of peptides was estimated using RSLCnano system online coupled with HCTUltra ion trap (Bruker Corporation), which is based on the area under total ion current curve using MEC cell line tryptic digest as an external calibrant.

25 μM protein samples were prepared in 10 mM ammonium acetate, pH 7.4. Methanol (10% v/v final concentration) was added to the protein samples immediately prior to infusion via a syringe pump at a rate of 250 mL h⁻¹. Positive-ion electrospray mass spectra of the mutant proteins (25 mM, 10 mM ammonium acetate, pH 7.4, 10% methanol) were acquired on either a Bruker Daltonics HCTultra Ion Trap or a Bruker Daltonics MicrOTOF mass spectrometer. Typical acquisition times were 1.5 min using an m/z range of 500–3000. Data were averaged, smoothed, baseline-subtracted and deconvoluted onto a true mass scale in Bruker Daltonics Compass DataAnalysis.

Microdissected tissue samples were lysed in SDT buffer (4% SDS, 0.1M DTT, 0.1M Tris/HCl, pH 7.6) in a thermomixer (Eppendorf ThermoMixer^® C, 30 min, 95°C, 750 rpm). After that, samples were centrifuged (15 min, 20,000 x g) and the supernatant used for filter-aided sample preparation as described elsewhere [46 (link)] using 0.5 μg/sample of trypsin (sequencing grade, Promega). Resulting peptides were used for LC-MS/MS analyses. Total peptide amount after sample preparation was estimated using LC-MS analysis on RSLCnano system online coupled with HCTUltra ion trap (Bruker Daltonics) based on the area under the total ion current curve, whereby MEC cell line tryptic digest was used as external calibrant. For final analyses, we used app. 2 μg of tryptic digests.