1290 infinity 2 lc system

Peptide samples were analyzed using Agilent 1290 Infinity II LC system coupled with Agilent 6545XT Q-TOF mass spectrometer. LC separation was conducted on AdvanceBio Peptide Mapping column (120 Å, 2.1 × 150 mm, 2.7 µm) at 60 °C. Injection volume was 10 µl. Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. LC gradient was set as follows; 0% B for 2 min, 0–20% B in 33 min, 20–30% B in 20 min, 30–50% B in 10 min, 50–90% B in 5 min, 90% B for 5 min, 90–0% B in 5 min and 0% B for 5 min, with constant flow rate of 0.4 ml min⁻¹. MS analysis was conducted in positive mode with a mass range of 100–1700 m/z. MS parameters were set as follows; gas temperature at 325 °C, nebulizer at 35 psi, dying gas at 13 L min⁻¹, sheath gas temperature at 350 °C, sheath gas flow at 12 L min⁻¹, capillary voltage at 4000 V, nozzle voltage at 500 V, fragmentor voltage at 175 V and skimmer voltage at 65 V. Acquisition time was 1 spectrum per s. Maximum 2 precursor ions per cycle were selected for MS/MS fragmentation. Collision energy (CE) was varied according to the charge state of the peptide. For peptides with charge + 1 and + 2, the CE was calculated using a formula of (3.1 × ((m/z)/100) + 1), while peptides with charge ≥  + 3, the CE was calculated using a formula of (3.6 × ((m/z)/100) − 4.8).

Peptides were analyzed using a Q Exactive^TM Hybrid Quadrupole-Orbitrap^TM Mass Spectrometer (Thermo Fisher Scientific), which was connected to a 1290 Infinity II LC System (Agilent). The trap column was made of C18 (Dr. Maisch Reprosil) material and the analytical column was a 50 cm, 50 μm inner diameter Poroshell C18 (Agilent) column. Both the trap and analytical columns were packed in-house. Solvent A consisted of 0.1% formic acid (Merck) in deionized water (Merck) and Solvent B of 0.1% formic acid in 80% acetonitrile (Biosolve). Peptides were first trapped at 50 μl/min with solvent A and then eluted with solvent B in a 120 min gradient at 100 nl/min: 0–10 min, 100% solvent A; 10.1–105 min, 13–40% solvent B; 105–108 min, 40–100% solvent B; 108–109 min, 100% solvent B; 109–110 min, 0–100% solvent A; 110–120 min, 100% solvent A. The Orbitrap was operated in a data-dependent manner, with the following settings: ESI voltage, 1700 V; inlet capillary temperature 320°C; full-scan automatic gain control (AGC) target, 3 × 10⁶ ions at 35000 resolution; scan range, 350–1500 m/z; Orbitrap full-scan maximum injection time, 250 ms; MS2 scan AGC target, 5 × 10⁴ ions at 17500 resolution; maximum injection, 120 ms; normalized collision energy, 25; dynamic exclusion time, 30; isolation window 1.5 m/z; 10 MS2 scans per full scan.

Lipid extracts were analysed on an Agilent 1290 Infinity II LC System coupled to the Agilent 6546 LC/Q-TOF system using a ZORBAX Eclipse Plus, C18, 95 Å, 1,8 µm, 2,1 × 50 mm (Agilent). Samples for MRM targeted analysis were measured with the same chromatographic system connected to the Agilent 6495 LC/TQ. The Agilent Data Acquisition Software Version 10.1 was used for all LC–MS analyses.
For the chromatographic runs, Water/Acetonitrile (6:4, v/v) + 10 mM Ammonium Formate was used as Mobile Phase A and Isopropanol/Acetonitrile (9:1, v/v) + 10 mM Ammonium Formate was used as Mobile Phase B, with an elution gradient starting at 0 min with 80% A and including: 2 min 40% A, 12 min 0% A, 14 min 80% A. The stop time was set at 15.8 min with a flow rate of 0.4 mL/min and a column temperature of 40 °C. An injection volume of 4 µL was used in positive mode and 8 µL in negative mode. For the dilution series, injection volumes of 0.25, 0.5, 1, 2, 4 and 8 µL were used.
The ion source parameters for DDA and DIA were as follows: Gas temperature 325 °C, Drying gas 10L/min, Nebulizer 20 psi, Sheath gas temperature 350 °C, Sheath gas flow 12L/min, Capillary voltage 3500 V, Nozzle voltage 1000 V, Fragmentor voltage 180 V, Skimmer voltage 45 V and Octopole RF 750 V.

Aliquots (40 µL for (positive mode) and 120 µL for (negative mode)) of thawed plasma (NIST SRM 1950 Metabolites in Frozen Plasma, Sigma, St. Louis USA) were each extracted using a modified Folch extraction procedure [36 (link)] and reconstituted in 100 µL of a methanol/chloroform mixture (9:1, v/v). LC separation was performed on an Agilent 1290 Infinity II LC System, with a 19 min gradient time on a reverse phase C18 column (Agilent InfinityLab Poroshell 120 EC-C18, 3.0 × 100 mm, 2.7 µm). Mobile phase consisted of 10 mM ammonium acetate and 0.2 mM ammonium fluoride in 9:1 water/methanol, while mobile phase B consisted of 10 mM ammonium acetate and 0.2 mM ammonium fluoride in 2:3:5 acetonitrile/methanol/isopropanol. Negative and positive polarity data was acquired on the Agilent 6546 LC/Q-TOF using iterative MS/MS acquisition mode on 6 injections of extracted plasma for each polarity [37 ]. Detailed experimental methods for chromatography and mass spectrometry can be found in Supplemental Table S1 and Table S2, respectively, and in the Agilent application note 5994-0775en [37 ]. Two methods were used, a high-load and a low-load method, to determine the effect of high injection volumes/concentration on the number of annotations using the Agilent 6546 LC/Q-TOF.