Micromass zq mass spectrometer

The TAG molecular species were identified with HPLC-APCI/MS. The analyses were performed in a solvent delivery system coupled to a Micromass ZQ Mass Spectrometer (Waters) fitted with an APCI source, with full-scan acquisition. Data acquisition, processing, and instrument control were managed with the Xcalibur™ software (Thermo Scientific). The instrumental conditions were vaporizer temperature 400°C, capillary voltage 6.0 kV, and corona voltage 40 V. The spectra were obtained over the range of m/z 80–2000, with a scan time of 1.0 s.

The electrospray ionization (ESI) mass spectra were recorded on a Waters/Micromass ZQ mass spectrometer. The measurements were performed for the solutions of LasGlu and Las X (5 × 10⁻⁴ mol dm⁻³). The samples were prepared in dry acetonitrile, and were infused into the ESI source using a Harvard pump, at a flow rate of 20 mL min⁻¹. For the standard ESI mass spectra, the cone voltage was 30 V. The source temperature was 120 °C and the desolvation temperature was 300 °C. Nitrogen was used as the nebulizing and desolvation gas, at flow-rates of 100 and 300 dm³ h⁻¹, respectively. The mass range for ESI experiments was from m/z = 100 to m/z = 1300.

The resulting fractions were analyzed by HPLC-DAD-MS, using a Waters 2695 equipment coupled to a Waters 2996 photodiode detector and to a Waters micromass ZQ mass spectrometer fitted with electrospray interface (ESI). The column was a Phenomenex Gemini C18 (250 × 2.0 mm, 5 μm particle size). Elution system: A solvent (3% methanol, 2% acetic acid, and 95% water) and B solvent (93% methanol, 2% acetic acid, and 5% water) under gradient conditions: 0 min 100% A, 5 min 95% A, 30 min 50% A, 40 min 50% A, 50 min 100% B. The column was re-equilibrated before the following injection. Prior to their injection into the HPLC equipment, the samples were filtered through a 0.22 µm pore-size membrane (Millipore, Burlington, MA, USA). The injection volume was 20 μL and the flow rate 0.2 mL min⁻¹. The following parameters were used for ESI-MS identification: positive and negative ionization modes with N₂ as drying gas at a flow of 11 mL min⁻¹, 250 °C drying temperature, 3500 V capillary voltage, and 15 V capillary exit. The m/z scanning range covered the 100–1000 uma interval. The injection volume was 20 μL.

Low-resolution electrospray ionization mass spectrometry (LR-ESI−MS) results were collected using a Waters micromass ZQ mass spectrometer (Waters, Milford, MA, USA). Nuclear magnetic resonance (NMR) was conducted using a Varian NMR spectrometer (¹H, 500 MHz; ¹³C, 125 MHz; Varian, CA, USA) with tetramethylsilane, and chemical shifts were displayed as δ values. High-performance liquid chromatography (HPLC) was subjected to a Waters system equipped with a 600 controller, UV detector (996 photodiode array), and Luna 5 μm C18(2) 100 Å column (250 mm × 10 cm, Phenomenex, CA, USA). Open column chromatography was performed with silica gel (Merck, Germany). For thin-layer chromatography (TLC) analysis, 60 F254 and RP-18 F254S plates (Merck, Germany) were used, and visualization of the plates was performed using UV light and 10% sulfuric acid solution.

All trivalent SLPs were synthesized on a Prelude peptide synthesizer (Protein Technologies, Inc., Tucson, AZ, USA) at a 40-μmol scale using standard Fmoc protocols. All amino acids were double coupled using 3-fold excess of Fmoc-amino acids relative to the Fmoc-amino acid alko-PEG resin (0.20–0.25 mmol/g, Watanabe Chemical, Hiroshima Japan). Fmoc deprotection was performed using 20% piperidine/NMP. Coupling was carried out using amino acid/HBTU/HOBt/DIEA (1:1:1:2) in NMP. NMP top washes (5 × 0.5 min) were performed between deprotection and coupling steps. One NMP top wash was used between the double couplings. After completion of couplings, one NMP top wash was performed before the next deprotection step. Cleavage was performed with TFA/H2O/thioanisole/EDT/EMS/thiophenol (82:5:5:3:2:3) plus 2-methylindole (10 mg/mL). Crude peptides were precipitated from cold ether and collected by centrifugation. The resulting crude peptides were purified with preparative reversed phase high performance liquid chromatography (HPLC) (Waters Corp., Manchester, UK) with an ODS column. All peptides were characterized using a Micromass ZQ mass spectrometer (Waters) equipped with an ESI source. Other peptides (epitope peptides, bivalent peptides) were synthesized and analyzed by Toray Research Center, Inc. (Tokyo, Japan).