Amazon speed etd

SynthesisCell™ (Antec Leyden B.V., Leiden, The Netherlands) was used to generate mg quantities of metabolites from primary bile acids, cholic acid, and chenodeoxycholic acid. The cell was equipped with a flat smooth magic diamond (BDD, 4.5 × 4.5 × 0.2 cm) working electode, a coiled platinum counter electrode, and a Pd/H₂ reference electrode. Electrochemical potential was again controlled using a Roxy™ potentiostat (Antec Leyden B.V., Leiden, The Netherlands). 80 mL of a solution containing 2 mM of the primary bile acids cholic acid or chenodeoxycholic acid (in 90% methanol (v/v) and 10% purified and double-distilled water (v/v) with 20 mM of ammonium formate) was filled into the glass reaction vessel. The temperature of the electrochemical cell during oxidation was 20 °C. After placing the synthesis cell on the magnetic stirrer, a constant potential (which was identified in Section 4.2) was applied. Oxidation was monitored by the manual collection of 500-µL aliquot samples taken in 30-min intervals and manual injection via syringe pump into an ESI-MS ion trap mass analyzer in positive ion mode (amaZon speed ETD, Bruker Daltonics, Bremen, Germany), with the following mass spectrometer settings: ion spray voltage: 4.5 kV; ion source heater: 350 °C; source gas: 55 psi. For the instrumental setup, see Figure 4.

The chemical analysis from TbHPE was performed by HPLC (LC-20AD, Shimadzu Corp, Kyoto, Japan) using a column Phenomenex Luna C-18 (250 × 4.6 mm, 5 µm) at 25 °C, with mobile phase ultrapure water containing 0.1% formic acid (A) and methanol (B), linear gradient applied: 0 min, 5% B; 1−60 min, 5%−100% B; 60−70 min, 100% B at flow of 1 mL/min. The liquid chromatography was coupled to a mass spectrometer (Amazon Speed ETD, Bruker, Massachusetts, USA) with electrospray ionization (ESI) and an ion-trap (IT) analyzer in negative mode, using the conditions: 4.5 kV and 325 °C capillary voltage and temperature, respectively, entrainment gas (N₂) flow 12 L/min, nitrogen nebulizer pressure at 27 psi. The acquisition range was m/z 100–1200, with more than two events.

Aliquots (15 μL) of the in-solution digested samples (TFE) were separated by nanoLC (Ultimate3000 nanoRSLC; ThermoFisher Scientific, Germering, Germany) operated in trap column mode (3 μm beads, 75 μm inner diameter, 2 cm length; ThermoFisher Scientific) equipped with a 25 cm separation column (2 μm beads, 75 μm inner diameter, ThermoFisher Scientific) and applying a linear 240 min gradient from 2% v/v to 50% v/v acetonitrile with subsequent re-equilibration (eluent A: 0.1% v/v formic acid; eluent B: 80% v/v acetonitrile, 0.1% v/v formic acid). The nanoLC effluent was continuously analyzed by an online-coupled iontrap mass spectrometer (amaZon speed ETD; Bruker Daltonik GmbH, Bremen, Germany) using an electrospray ion source (Captivespray; Bruker Daltonik GmbH) operated in positive ion mode as described in detail before (Wöhlbrand et al., 2016 (link)).
In case of SDS-PAGE separated samples, the entire sample lane was cut into eight pieces, which were further cut into small pieces of ∼1–2 mm² and subjected to in-gel digest as described by Kossmehl et al. (2013) (link). Generated peptides were analyzed by nanoLC ESI-iontrap MS/MS (setup see above) using a linear 130 min gradient from 2% v/v to 50% v/v acetonitrile with subsequent re-equilibration.

The pollen extract was analyzed by HPLC (LC–20AD Shimadzu, Kyoto, JP) and a Phenomenex Luna C–18 (250 × 4.6 mm-5 µm) column at 25 °C was used. The mobile phases consisted of ultrapure water containing 0.1% formic acid (A) and methanol (B). The following linear gradient was applied: 0 min, 5% B; 1−60 min, 5−100% B; 60−70 min, 100% B at flow of 1 mL/min. The LC was coupled to a mass spectrometer (Amazon Speed ETD, Bruker, Massachusetts, USA) equipped with electrospray ionization (ESI) and an ion–trap (IT) type analyzer in negative mode, under the following conditions: 4.5 kV capillary voltage, capillary temperature 325 °C, entrainment gas (N₂) flow 12 L/min, nitrogen nebulizer pressure at 27 psi. The acquisition range was m/z 100–1000, with two or more events.

High-performance gel permeation chromatography (HPGPC) was used to assess weight-averaged molecular weights of the enzyme products with an LC-10Avp system (Shimadzu Company, Japan) and a TSK-gel G-3000 PWXL column (7.8 × 300 mm). The UPLC-MS method was performed using a Waters Acquity H-Class UPLC system linked to an ESI-MS mass spectrometer (amaZon speed ETD, Bruker, Germany) in the negative ion mode. Operating parameters were as follows: capillary voltage, 4.5 KV; capillary temperature, 200°C; nebulizer gas, 2 bar; dry gas, 6 l/min; scan range, 100–1000. RG oligosaccharide separation was carried out on an Acquity UPLC BEH Amide column (1.7 μm, 2.1 mm × 150 mm) at 35°C with a flow rate of 0.3 ml/min mobile phase. The mobile phase consisted of ACN and H₂O in a ratio of 20/80 (v/v) for mobile phase A, 80/20 (v/v) for mobile phase B, and pH 3.0 200 mM ammonium formate/50 mM formic acid buffer for mobile phase C. The run time for oligosaccharide separation was 60 min, and the elution procedure was as follows: concentration of C remained at 5% during the entire elution process for 0–30 min, 0%-20% A; 30–31 min, 20–35% A; 31–40 min, containing 35% A; 40–41 min, 35–0% A; 41–50 min, containing 0% A.

COOH-terminal peptides of CXCL4 [CXCL4(47-70)] and CXCL9 [CXCL9(74-103), CXCL9(74-93), CXCL9(86-103)] were chemically synthesized using fluorenyl methoxycarbonyl (Fmoc) chemistry using an Activo-P11 automated synthesizer (Activotec, Cambridge, UK), as previously described (45 (link)). Part of the material was site-specifically biotinylated or fluorescently labeled at the NH₂-terminus using biotin-p-nitrophenyl ester (Novabiochem, Darmstadt, Germany) or 5(6)-carboxytetramethylrhodamine (TAMRA; Merck Millipore, Darmstadt, Germany), respectively (42 (link)). After synthesis, intact synthetic peptides were purified and identified by mass spectrometry (Amazon SL or Amazon Speed ETD ion trap, Bruker, Bremen, Germany).

Tryptic digestion of alpha-lactalbumin was performed with an aqueous solution of alpha-lactalbumin (Sigma Aldrich Chemie GmbH, Steinheim, Germany) in a concentration of 1 mg/mL protein (in purified and double-distilled water). It was digested tryptically (trypsin from porcine pancreas; Sigma Aldrich Chemie GmbH, Steinheim, Germany) for 16 h at 37 °C (protein-enzyme ratio 100:1). Digestion samples were used after SPE cleaning procedure via RP18ec SPR cartridges (Macherey Nagel GmbH & Co. KG, Düren, Germany) with 60% ACN for conditioning and washing steps and 0.2% aqueous formic acid for equilibration and elution steps. Afterwards, samples were dried using gaseous nitrogen and re-dissolved in a defined volume using 0.2% aqueous formic acid. Tryptic digest was analyzed using ESI-MS ion trap mass analyzer in negative ion mode (amazon speed ETD, Bruker Daltonik GmbH, Bremen, Germany), with following mass spectrometer settings: ion spray voltage: 4.5 kV; ion source heater: 350 °C; source gas: 55 psi. An assignment of signals and a resulting identification of the peptides were performed using the UniProtKB database (http://www.uniprot.org/) and the SIB Bioinformatics Resource Portal ExPASy (https://www.expasy.org/). Mentioned databases provide a comparison with the theoretical tryptic digestion of sequences. The peptides detected during this analysis are shown in Table 1.