Lc msd tof

Protein concentration was determined by measuring the absorbance at 280 nm and using the following theoretical molar extinction coefficients determined with the Expasy ProtParam tool (http://web.expasy.org/protparam/): MBP-CXCL12-Strep (80580 M⁻¹.cm⁻¹), MBP-CXCL12-LT-Strep (94560 M⁻¹.cm⁻¹), MBP-CCL5-Strep (85050 M⁻¹.cm⁻¹), MBP-CCL5-LT-Strep (99030 M⁻¹.cm⁻¹), CXCL12-HIS (8730 M⁻¹.cm⁻¹), CXCL12-LT-HIS (22710 M⁻¹.cm⁻¹), CXCL12-Strep (14230 M⁻¹.cm⁻¹), CXCL12-LT-Strep (28210 M⁻¹.cm⁻¹), CCL5 (13200 M⁻¹.cm⁻¹), CCL5-Strep (18700 M⁻¹.cm⁻¹), CCL5-LT-Strep (32680 M⁻¹.cm⁻¹). CXCL12-HIS (20 µM in 20 mM sodium phosphate, pH 6.0) and CXCL12-LT-HIS (20 µM in 20 mM sodium phosphate, pH 8.0) circular dichroism (CD) spectra were measured at 25°C in a Jobin Yvon CD6 spectropolarimeter over a 190- to 260-nm range (1-nm interval) in a 0,1 cm optical path length quartz cuvette. Three repetitive scans were taken for each sample and corrected for solvent contributions. Molar ellipticity was calculated as previously described [13] . Protein masses were determined using an electrospray TOF mass spectrometer (Agilent, LC/MSD TOF) directly coupled with the HPLC system (Agilent 1100 series).

¹H-NMR and ¹³C-NMR spectra were recorded with a Varian Mercury 400 or 500 spectrometer using tetramethylsilane as the internal standard in methanol-d₄, DMSO-d₆, or chloroform-d. High-resolution mass spectrometry (HRMS) data were measured on a Thermo Exactive Orbitrap Plus spectrometer. Liquid chromatography–mass spectrometry (LC-MS) was conducted on an Agilent 1100 series HPLC and an Agilent LC/MSD TOF. All of the solvents and chemicals were purchased from commercial sources: Sigma-Aldrich Chemical Co., Beijing Ou-he Reagents Co., Beijing Shiji-Aoke Biotechnology Co., and Shanghai Jingke Chemistry Technology Co. with a purity of more than 95% (LC-MS). All chemicals and solvents used were of reagent grade without further purification or drying before use. All the reactions were monitored by thin-layer chromatography (TLC) under a UV lamp at 254 nm. Column chromatography separations were performed using silica gel (200–300 mesh).

All air sensitive manipulations were carried out under a dry argon or nitrogen atmosphere. THF and CH₂Cl₂ were dried using a column solvent purification system. Analytical thin-layer chromatography was performed on SiO₂ (silica gel 60A 35–70 μm, Carlo Erba, Val de Reuil Cedex, France), and the spots were located with 1% aqueous KMnO₄. Chromatography refers to flash chromatography and was carried out on SiO₂ (SDS silica gel 60 ACC, 35–75 mm, 230-240 mesh ASTM). NMR spectra were recorded at 300 or 400 MHz (¹H) and 100.6 MHz (¹³C), and chemical shifts are reported in δ values downfield from TMS or relative to residual chloroform (7.26 ppm, 77.0 ppm) as an internal standard. Data are reported in the following manner: chemical shift, multiplicity, coupling constant (J) in hertz (Hz), integrated intensity, and assignment (when possible). Assignments and stereochemical determinations are given only when they are derived from definitive two-dimensional NMR experiments (HSQC-COSY). IR spectra were performed in an Avatar 320 FT-IR spectrophotometer (Thermo Nicolet, Madison, WI, USA) and only noteworthy IR absorptions (cm⁻¹) are listed. High resolution mass spectra (HMRS; LC/MSD TOF, Agilent Technologies, Santa Clara, CA, USA) were performed by Centres Científics i Tecnològics de la Universitat de Barcelona.

CE-TOFMS and LC-TOFMS-based metabonomic profile of frozen worm pellets was performed (Human Metabolome Technologies, Yamagata, Japan). The wild type N2, DR treatment, or eat-2 mutant one-day-old animals were raised over a period of 4 days. Frozen worm samples were mixed with 800 µl of 50% acetonitrile (v/v) containing internal standards (20 µM for cation measurement and 5 µM for anion measurement), homogenized in the presence of beads, and centrifuged. The supernatant was then filtered through 5-kDa cut-off filters (Ultrafree-MC PLHCC, Merck KGaA, Darmstadt, Germany) to remove macromolecules. The filtrate was centrifugally concentrated and diluted in 25 µl of ultrapure water for CE/TOF-MS analysis. For LC/TOF-MS analysis, frozen worm samples were mixed with 500 µl of 1% formic acid in acetonitrile (v/v) containing internal standards, homogenized, and centrifuged. The supernatant was filtered through a 3-kDa cut-off filter (Nanocep 3 K Omega, Pall Corporation, MI, USA) to remove proteins and then passed over a column (Hybrid SPE phospholipid 55261-U, Supelco, PA, USA) to remove phospholipids. The column eluate was desiccated and suspended in 100 µl of 50% isopropanol. Three independently collected worm pellets were used for each assay with CE-TOF/MS system and 1200 series Rapid Resolution system SL, subsequently LC/MSD-TOF (Agilent Technologies, CA, USA).