Series 10ad vp lc system

Plasma sample preparation and analysis by LC-MS/MS were performed as previously described^{24 (link)}. Briefly, AGT was selectively extracted from plasma (50 µL) using 2-dimensional chromatography employing Concanavalin A lectin affinity and reversed-phase steps. Enriched AGT samples were deglycosylated using PNGase F followed by differential alkylation of the reduced and the oxidized form of AGT using ¹³C₀,D₀- and ¹³C₂,D₂-iodoacetamide respectively. Protein digestion was carried out using sequencing grade chymotrypsin. Peptides were analysed by a Shimadzu series 10AD VP LC system (Shimadzu, Columbia, MD) using a C18, 300 Å, 100 × 1 mm, 3 µm column (ACE, Reading, UK) and a mobile phase of water and acetonitrile both with 0.1% formic acid. Chymotrypsin-produced signature peptides were characterized using a 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (SCIEX, Foster City, CA, USA). Multiple reaction monitoring (MRM) transitions were monitored for the following AGT signature peptides: (1) AGT marker peptide (SVTQVPF), which was used to infer the plasma level of total AGT; (2) ¹³C₀,D₀-iodoacetamide alkylated Cys18 peptide (HLVIHDESTC¹⁸EQL), which corresponds to the reduced form of plasma AGT and (3) ¹³C₂,D₂- iodoacetamide alkylated Cys18 peptide (HLVIHDESTC¹⁸EQL), which corresponds to the oxidized form of plasma AGT.

Concentrations of 34 oxylipins (see S1 Table) were quantified by liquid chromatography–mass spectrometry (LS-MS/MS) using a validated quantitative method based on that described by Wong et al.[21 (link)]. Lipids not included in [21 (link)] were measured using the following LC-MS/MS settings (precursor / product ions / collision energy): 17R resolvin D1 (375.3 / 141.0 / 22), 6-ketoPGF1a (369.1 / 162.8 / 36), resolvin D2 (375.3 / 175.0 / 30), 17-HDoHE (343.1 / 343.1 / 9). The HPLC system used was a Shimadzu series 10AD VP LC system (Shimadzu, Columbia, MD, USA) and the MS system used was an Applied Biosystem MDS SCIEX 4000 Q-Trap hybrid triple-quadrupole–linear ion trap mass spectrometer (Applied Biosystem, Foster City, CA, USA) equipped with an electrospray ionisation (ESI) interface. Quantification of the eicosanoids was calculated using fully extracted calibration standards for each of the analytes. Before quantification of lipid-derived inflammatory mediators of rat synovial fluid, blank filter papers were stained with equivalent concentrations of calibration standards (100pM, 500pM, 1nM, 2.5 nM, 5nM, 10nM) and were compared with actual standard calibration curves. Quantification was performed using Analyst 1.4.1. Identification of each compound in plasma samples was confirmed by LC retention times of each standard and precursor and product ion m/z ratios.

The HPLC system used was a Shimadzu series 10AD VP LC system (Shimadzu, Columbia, MD). The HPLC column used was ACE C18 (150 × 2.1 mm ID 3 µm particle size) with a guard column (Security Guard Cartridges ACE 3 C18 for ID 150 × 2.1 mm column). Mobile phase A was 0.02% formic acid in methanol:acetonitrile (1:4, v/v); mobile phase B was 0.02% formic acid in 100% water. The starting flow rate was 200 µl/min. Strata-X polymeric SPE column (200 mg/6 ml) was purchased from Phenomenex, Macclesfield, UK. The evaporator used was a Jouan centrifugal evaporator (Saint-Herblain, France). The MS system used was an Applied Biosystem MDS SCIEX 4000 Q-Trap hybrid triple-quadrupole-linear ion trap mass spectrometer (Applied Biosystem, Foster City, CA) equipped with an ESI interface.
Standards for all compounds were purchased from Cayman Chemicals (Ann Arbor, MI).
One batch of blank human plasma (for the OA case-control) or serum (for the TwinsUK study) acted as an analytical quality control used to confirm the day-to-day accuracy/precision of the method during the analysis of each batch of sample analysis.