Ultimate 3000 autosampler

Phosphopeptides eluted from IMAC were subjected to an offline HILIC fractionation method as previously described (Albuquerque et al. 2008 (link)). Fractionated samples were run on an LTQ Orbitrap XL using a 1100 Quad PUMP HPLC system (Agilent, Santa Clara, CA) with Ultimate 3000 autosampler (Dionex, Sunnyvale, CA).

10 mg of samples was weighed and tetrahydrofuran (THF) containing 5% of toluene (internal standard) was added in order to reach a concentration of 5 mg/mL. After 5 min of shaking, samples were filtered on 0.45 µm GHP microfilters (PALL) prior to analysis by size-exclusion chromatography (SEC). SEC analyses were performed using THF stabilized with BHT as the eluent at 1 mL/min (Ultimate 3000 Pump, Dionex). 10 μL was injected (Ultimate 3000 Autosampler, Dionex) on either a PLgel Mixed C column (5 μm, 7.5 mm × 600 mm, Agilent) in the case of DHPs, or a PLgel Mixed E column (3 μm, 7.5 mm × 600 mm, Agilent) in the case of lignins, and the signal was observed at 280 nm (Ultimate 3000 UV/vis detector, Dionex). The molar mass distributions were determined using a calibration curve based on 10 polystyrene standards ranging from 580 to 364,000 g/mol (Agilent) for the Mixed C column (Supplementary Figure S1) or from 162 to 22,290 g/mol (Agilent) for the Mixed E column (Supplementary Figure S2).

Tryptophan (TRP), L-kynurenine sulfate (KYN), and kynurenic acid (KYNA) were obtained from Sigma-Aldrich, Co. (St. Louis, MO, USA) and used as a standard. HPLC-grade chemicals were purchased from J.T. Baker Chemicals (Aventor Performance Materials Poland S.A., Gliwice, Poland) or Sigma-Aldrich. TRP, KYN, and KYNA were analyzed by high-performance liquid chromatography (HPLC), according to the protocol of Zhao et al. (2010) [56 (link)], with minor modifications. Briefly, each serum sample was added 6% HClO₄ and centrifuged at 12,000× g for 30 min at 4 °C. The resulting supernatant was applied to an HPLC system consisting of the Dionex P680 Pump, UltiMate 3000 Autosampler with column compartment, RS Variable Wavelength UltiMate 3000 Detector, and RF 2000 Fluorescence Detector (Dionex, Sunnyvale, CA, USA). An Agilent HC-C18 column (250 × 4.6 mm i.d.; 5-µm particle size) was used. The mobile phase was composed of 20 mM sodium acetate, 5 mM zinc acetate, and 4% acetonitrile; the flow rate was 1.0 mL/min. Detectors were set at wavelengths 250 nm for TRP, 365 nm for KYN, and at excitation wavelength of 348 nm and emission at 398 nm for KYNA determination. Control of the HPLC system and data analysis were performed with the Chromeleon software (Dionex^TM Chormeleon^TM version 7.2.6 (10049), serial number 117836).