Ltq orbitrap velos instrument

Urine sample preparation for LC-MS analysis was performed as we previously reported19 (link). Briefly, the urine samples were thawed at room temperature. 100 μl of each thawed urine sample was precipitated by 100 μl of methanol. The mixture was then centrifuged under 14000 g for 10 minutes at 4 °C, and the supernatant was used for LC-MS analysis.
Each 10 μL aliquot of extract was injected into a Shimadzu Prominence LC system (Shimadzu) coupled online to an LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, MA, USA) set at 30000 resolution (at m/z 400). Both positive and negative ion modes were used for sample analysis. The mass scanning range was 50–1000 m/z and the capillary temperature was 350 °C. Nitrogen sheath gas was set at a flow rate of 30 L/min. Nitrogen auxiliary gas was set at a flow rate of 10 L/min. Spray voltage was set to 4.5 kV and 3.0 kV for positive or negative ion mode, respectively. The LC-MS system was run in binary gradient mode. Solvent A was 0.1% (v/v) formic acid/water and solvent B was 0.1% (v/v) formic acid/methanol. The flow rate was 0.2 ml/min. A C-18 column (150 × 2.1 mm, 3.5 μm, Agilent, USA) was used for all analysis. The linear gradient was as follows: 5% B at 0 min, 5% B at 5 min, 100% B at 8 min, 100% B at 9 min, 5% B at 18 min and 5% B at 20 min.

For each LC-MS/MS run, 10 nmol of the reaction mix from the previous section dissolved in 10 µL 0.1% formic acid was used. Samples were loaded using the autoinjector unit of a nanoAcquity^TM ultra performance LC system (Waters). Each sample was first loaded onto a pre-column (nanoAcquity 10K 2G V/M trap column, C18, 5 μm, 180 μm × 20 mm) at a flow rate of 10 µL/min. Peptides were then separated using a separating column (Nikkyo Technos Co. Ltd., Tokio, Japan, C18, 5 μm, 100 μm × 150 mm) at a flow rate of 0.4 μL/min. Separations were performed at 20 °C and the pressure range was 900–1000 psi. The composition of solvent A was aqueous 0.1% formic acid and the composition of solvent B was 0.1% formic acid in acetonitrile (ACN, product number 900667). The solvent gradient was 1% B (0–1 min), 1–60% B (1–15 min), 60–90% B (15–15.01 min), 90% B (15.01–20 min), 1% B (20–30 min). MS/MS analysis was performed using an LTQ Orbitrap Velos instrument (Thermo Fisher, Waltham, MA, USA). The MS1 m/z scan range was 300–2000 Da. The 20 most abundant precursor ions from each MS scan were isolated for MS2 analysis and fragmented via CID with helium gas using a collision energy setting of 35. Fragmentation analysis occurred in the linear ion trap with a mass accuracy window of ±0.8 Da.

The peptides were injected into the loop of an Eksogent nano LC-ultra 1D plus HPLC system equipped with a C18 column (200 mm home-made Altima C18 analytical column, 100 μm ID 3 μm particle size). Peptides were separated using a linear gradient of 5% solvent A (0.1% acidic acid, 5% ACN) and 45% solvent B (0.1% acidic acid, 80% ACN) in 50 min. The LC system was directly coupled inline with an LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific). The LTQ-Orbitrap was set to data-dependent mode to switch automatically between MS and MS/MS. MS spectra range from 330 until 2000 m/z can be acquired in the Orbitrap at an FWHM resolution of 30,000 after accumulation to 500,000 in the linear ion trap with one microscan. The five most abundant precursor ions were selected for fragmentation by CID with an isolation width of 2 DA. CID was performed in the linear ion trap after accumulation to 50,000 with 1 microscan.