Omix c18 100 μl tips

Total proteins from S. elongatus were extracted, as described [22 (link)]. Total protein extracts (4 mg/IP) were immunopurified using anti-GFP antibody-coupled magnetic beads of 50 nm (MACS^® Technology, Miltenyi, Bergisch Gladbach, Germany), digested in column with trypsin and analyzed in a single run on the mass spectrometer [23 (link)]. The resulting tryptic peptide mixture was desalted prior to LC-MS/MS analysis on a C18 ZipTip (Omix C18 100 μL tips, Varian, Santa Clara, CA, USA), and the purified peptide mixture was analyzed by LC-MS/MS using a nanoflow RP-HPLC (LC program: linear gradient of 3–40% B in 100 min, solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile), which was on-line coupled to a linear ion trap Orbitrap (Orbitrap-Fusion Lumos, Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer operating in positive ion mode. Data acquisition was carried out in a data-dependent fashion, and the 20 most abundant, multiply charged ions were selected from each MS survey for MS/MS analysis (MS spectra were acquired in the Orbitrap, and CID spectra were acquired in the linear ion trap).

Plasma samples from a total of 376 subjects (see above for details and ethical approvals), from two independent cohorts, were analyzed via DI-FT-ICR MS (24 (link), 41 (link)). Before analyses, the metabolites (from 50 μl of blood plasma) were extracted by C18 solid-phase extraction (SPE) technology, using Omix C18 100 μl tips (Varian) and following the protocol described by Forcisi et al. (24 (link)). The extracts, diluted in methanol by a factor of 50, were analyzed in positive ESI mode via DI-FT-ICR MS, using a Bruker SolariX instrument equipped with a 12-T magnet (Bruker Daltonik GmbH, Bremen, Germany). The instrument was externally calibrated by injecting a solution of arginine (10 μg/ml) and observing corresponding peaks with m/z values equal to 175.11895 [M+H]⁺, 349.23062 [2M+H]⁺, 523.34230 [3M+H]⁺, and 697.45397 [4M+H]⁺. In the experiment, the flow rate of infusion was set to 120 μl/hour. Four hundred scans, each corresponding to 4 MWs in the interval from 147.4 to 1000.0 m/z, were acquired and averaged. The time of accumulation ion was set to 0.7 s, and the time of flight to the detector was set to 1 ms. The voltages of capillary and spray shields were set to 3800 and −500 V, respectively. The flow rate of nebulizer gas was kept at 2.2 bar, and the drying gas flow rate was set to 4 liters/min (at a temperature of 180°C).