Reprosil pur c18 aq column

Blood plasma was mixed with the two–fold volume of acetone/water (4/1; v/v), precipitated proteins were removed by centrifugation, and the supernatants were concentrated and analysed by a semi–preparative RP–HPLC (Reprosil–Pur C18–AQ column (150 × 10 mm; particle size: 10 µm) from Dr. Maisch HPLC GmbH (Ammerbruch; Germany). Elution was obtained with a MeCN/20 mM NH₄OAc_aq gradient (pH 6.8) as follows: 0–5 min 18% MeCN, 5–20 min up to 90% MeCN, 20–22 min 90% MeCN, 22–23 min down to 10% MeCN, 23–30 min isocratic 10% MeCN) at a constant flow rate of 3 mL/min.

Peptides were separated by Reprosil-Pur C18-AQ column (3μm; Dr. Maisch GmbH, Germany) using Easy nano-LC HPLC (Proxeon, Odense, Denmark). The HPLC gradient was 0–34% B solvent (A = 0.1% formic acid; B = 90% ACN, 0.1% formic acid) in 70 min at a flow of 250 nL/min. The MS analysis was performed using the LTQ-Orbitrap Velos (Thermo Scientific, Bremen, Germany). The mass range was 400–1500 m/z at a resolution of 30,000 at 400 m/z for a target value of 1e⁶ ions. For each MS scan, collision induced dissociation (CID) fragmentation was performed on the 20 most intense ions in the linear iontrap. The parameters for data acquisition were: activation time = 15 ms, normalized energy = 35, Q-activation = 0:25, exclusion = available with repeat count 1, exclusion duration = 30s and intensity threshold = 30.000, target ions = 2e⁴ [25 (link)]. All raw data have been submitted to PRIDE archive (https://www.ebi.ac.uk/pride/archive/), project accession: PXD008088.

Sample preparation and analysis of curcuminoids were previously reported in detail [10 (link), 11 (link)]. Briefly, one mL fluid was acidified with 10 μL 6 M hydrochloric acid and incubated with 100 μL beta-glucuronidase type H1 from Helix pomatia (1 mg/100 μL in 0.1 M sodium acetate buffer, Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) for 45 min at 37 °C. After triplicate extraction with 95 % ethyl acetate and 5 % methanol (v/v), supernatants were evaporated to dryness and resuspended in 150 μL methanol, vortexed for 20 s, stored in the dark for 10 min, vortexed for 20 s and transferred to HPLC vials. Twenty μL of each sample was injected into the HPLC system.
Curcuminoids were quantified on a Jasco HPLC system (Jasco GmbH, Gross-Umstadt, Germany) with a fluorescence detector (excitation wavelength 426 nm, emission wavelength 536 nm) and separated on a Reprosil-Pur C18-AQ column (150 mm × 4 mm, 3 μm particle size; Dr. Maisch GmbH, Ammerbuch, Germany) maintained at 40 °C. The mobile phase consisted of 52 % de-ionized water (adjusted to pH 3 with perchloric acid), 34 % acetonitrile and 14 % methanol. Curcuminoids were quantified against external standard curves (curcumin, purity ≥97.2 %, CAS # 458–37-7; DMC, purity ≥98.3 %, CAS # 22,608–11-13; BDMC, purity ≥99.4 %, CAS # 24,949–16-; Chromadex, Irvine, USA).

Samples were suspended in an appropriate buffer and analysed on a Q Exactive HF-X mass Spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) coupled with a high-performance liquid chromatography system (EASY nLC 1200, Thermo Fisher). Redissolved dried peptide samples were loaded onto the 150 μm by 2 cm ReproSil-Pur C18-AQ column (3 μm; Dr. Maisch) in Solvent A (0.1% formic acid in water), with a maximum pressure of 280 bar using Solvent A. Separation was then performed on a home-made 100 μm by 15 cm silica microcolumn using mobile phase B with a gradient of 4–100% (0.1% Formic acid in 80% ACN) at a flow rate of 600 nl/min for 75 min. Mass spectrometry was conducted under a data-dependent acquisition mode after the elution of peptides. The orbitrap instrument was used to conduct the MS1 full scan by scanning 300–1400 m/z at120,000 resolution. The maximal ion injection time was 80 ms with an automatic gain control (AGC) of 3e6. A top-speed MS2 acquisition was performed and selected precursor ions were subjected to higher energy collision dissociation (HCD) with 27% normalized collision energy. AGC at 5e4 was applied to analyze fragment ions. A maximum ion injection time was achieved by MS2 of 20 ms, while and the dynamic exclusion was 12 s. Data acquisition was performed with Xcalibur software (Thermo Scientific).

Peptides were resuspended in 0.5% acetic acid and separated by reverse phase liquid chromatography on an in-house packed 17-cm × 75-μm Reprosil-Pur C18-AQ column (1.9 μm, Dr. Maisch) using an EASY nLC-II nanoHPLC (Proxeon). The HPLC gradient was 0–40% solvent B (solvent A, 0.5% acetic acid; solvent B, 90% acetonitrile, 0.5% acetic acid) over 90 min at a flow of 250 nl/min. MS was performed using an LTQ-Orbitrap Velos Pro (Thermo Scientific). An MS scan (300–1750 m/z; MS AGC 3 × 10⁶) was recorded in the Orbitrap set at a resolution of 60,000 at 400 m/z followed by data-dependent collision-induced dissociation MS/MS of the 20 most intense precursor ions. An MS scan (300–1750 m/z; automatic gain control 3 × 10⁶) was recorded in the Orbitrap set at a resolution of 60,000 at 400 m/z followed by data-dependent collision-induced dissociation MS/MS of the 20 most intense precursor ions. Parameters for collision-induced dissociation were as follows: normalized energy 35, dynamic duration 60 s, maximum injection time 150 ms, and tandem MS automatic gain control 4 × 10⁵.