Agilent 1290 lc

The KS sample was completely degraded by enzymatic hydrolysis. KS (100 µg) was dissolved in 100 μL of digestion buffer containing 50 mM ammonium acetate (pH 7.0). Excess keratanase II (50 mU) was added to KS sample and incubated at 37 °C overnight with gentle agitation. Enzymatic digestion was terminated by heating in a 100 °C water bath, and then spun down at 12,000 rpm for 5 min; supernatant was used directly for HILIC-FTMS analysis.
HILIC-FTMS analysis was performed on an Agilent 1290 LC ultra-performance liquid chromatography (UPLC) system (Agilent Technologies, Wilmington, DE, USA) equipped with a LTQ ORBITRAP XL mass spectrometer (Thermo, Scientific, Waltham, MA, USA). The KSO were separated by a Luna HILIC column (150 × 2.00 mm, 3 μm, Phenomenex) at 25 °C. The mobile phase was a mixture of 5 mM NH₄OAc/98% acetonitrile (solvent A) and 5 mM NH₄OAc/H₂O (solvent B) at a flow rate of 150 μL/min. The gradient was programmed as 92% A initially and then linearly changed to 60% A over 58 min. The analysis was performed in the negative ion mode using a capillary temperature of 275 °C. The spray voltage was 4.2 kV and Nitrogen dry gas flowed at 40 L/min. Data acquisition and analysis were performed using Xcalibur 2.0 software (Thermo, Scientific, Waltham, MA, USA) and GlycReSoft 1.0 software (Publicly archived, http://code.google.com/p/glycresoft/downloads/list).

In this experiment, chromatographic separation was performed using an ACQUITY UPLC HSS T3 column (2.1 × 100 mm I.D., 1.8 μm, Waters, USA). The column was maintained at a temperature of 35 °C, and the mobile phases consisted of water (Phase A) and acetonitrile (Phase B) with the addition of 0.1% formic acid. The gradient program involved the application of a 5% solution of Phase B for the first 3 min, followed by a linear increase from 5 to 95% solution B over the next 12 min (3–15 min), and then maintaining the 95% solution B for an additional 2 min (15–17 min). A 5-minute post-treatment period was conducted with a flow rate of 350 µL/min. The sample injection volume was approximately 2 µL.
The data in this study were collected using Agilent 1290 LC and 6538 Q-TOF mass spectrometers, employing electrospray ionization in both positive and negative ionization modes. The drying gas N2 was delivered at a rate of 9 L/min, while the temperature was maintained at 360 °C. The nebulizer pressure was set at 39 psi, and the capillary voltage was adjusted to 4000 V and 3900 V for positive and negative ionization modes, respectively. The scan range for the mass spectra was set from 50 to 1000 m/z. To ensure accuracy and reproducibility, a reference solution was utilized for real-time correction of the mass spectra, with the lock mass (m/z 922.009798, 121.050873) serving as a reference point.

Metabolite analysis was performed by LC–MS, using hydrophilic interaction (HILIC) LC and high resolution QTOF mass spectrometry. Sample extracts (10 μL) were injected onto an Agilent 1290 LC fitted with a ZIC-pHILIC column (5 μm, 2.1 × 150 mm; Merck), and 20 mM ammonium carbonate (A) and acetonitrile (B) as the mobile phases. A 14 min gradient starting from 90% B to 40% B over 12 min, held for 2 min followed by washing at 5% B for 3 min and re-equilibration at 90% B, was used. Mass spectrometry utilized an Agilent 6545 QTOF with heated electrospray source operating in negative ionization mode and scan range m/z 50–1700. Conditioning was performed before each batch using 2–3 blanks and 5 mixtures of authentic standards (234 metabolites), which were analyzed in data-dependent MS/MS mode to facilitate downstream metabolite identification where necessary. PBQC samples were analyzed periodically throughout the analysis.

4-hydroxynonenal (4-HNE) was measured by GC/MS in selected ion monitoring (SIM) mode, with the O-PFB-oxime or O-PFB-oxime-TMS derivatives using benzaldehyde-D6 as an internal standard [101 (link)]. Aldehyde was analyzed using a 7890A GC–7000 quadrupole MS/MS (Agilent Technologies, Palo Alto, CA, USA) equipped with an HP-5ms capillary column (30 m length, 0.25 mm internal diameter). Target ions with an m/z 333.0 and 181.0 for 4-HNE-PFB-TMS and m/z 307.0 for IS derivatives were selected. The obtained results were normalized and are presented for milligrams of protein.
The 8-Isoprostaglandin F2 (8-isoPGF2) level was measured using LC-MS analysis performed on an Agilent 1290 LC coupled with an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) in negative multiple reaction mode [102 (link)] and the transition m/z 353→193 was selected. SEP-PAK C18 columns were used for sample purification.