Orbitrap fusion lumos ms

An EASY-nLC 1200 LC system (Thermo Fisher Scientific) interfaced
via a nanoSpray Flex ion source to an Orbitrap Fusion Lumos MS (Thermo
Fisher Scientific) was used for MS and MS/MS analysis. A single analytical
column setup using PicoFrit Emitters (New Objectives, 75 μm
in inner diameter) custom-packed with a Reprosil-Pure-AQ C18 phase
(1.9 μm in particle size and 19–21 cm in column length)
was applied in nLC. Two microliters of each sample was injected onto
the column, followed by elution with a gradient of solvent B from
3 to 32% at 200 nL/min for 45 min (solvent A: 100% H₂O
+ 0.1% (v/v) formic acid; solvent B: 80% acetonitrile + 0.1% (v/v)
formic acid). With the nominal resolution setting of 120,000, precursors
of MS1 scan (m/z 350–2000) were obtained.
Then, HCD-MS2 of the five most abundant multiply charged precursors
in the MS1 spectrum was acquired at the nominal resolution setting
of 120,000. To trigger data-dependent fragmentation events, the minimum
MS1 signal threshold was 50,000. Targeted MS/MS analysis was performed
by setting up a targeted MSn (tMSn) scan.

The liver dialysates obtained by OFM sampling were analyzed using ultra performance liquid chromatography-electrospray high resolution MS/MS (UPLC-ESI HR MS/MS). In brief, 80 μL dialysate sample was dried with a vacuum dryer, and then re-dissolved in 50 μL of ethanol containing 0.25 μg/mL isoprenaline (Sigma-Aldrich, USA) as internal standard (IS). The mixture was centrifuged for 15 min at a rate of 10,000g at 4 °C. The supernatant was collected for UPLC-ESI HR MS/MS analysis. Samples were loaded into the UPLC system (Ultimate 3000, Thermo Fisher Scientific, USA) equipped with a BEH amide column (1.7 μm, 2.1 mm ID × 20 mm, Waters, USA). The mobile phase A was water containing 20 mM ammonium acetate and the mobile phase B was acetonitrile containing 0.1% formic acid. The LC separations were 30 min per sample with a flow rate at 0.3 mL/min using LC gradient reported in the previous literature [24 (link)]. The metabolite profile was acquired using Orbitrap Fusion Lumos MS (Thermo Fisher Scientific, USA) with positive-ion mode. Major operating parameters were as follows: electrospray voltage + 3000 V, m/z range 150–1000, ion transfer tube temperature 325 °C, vaporizer temperature 275 °C, sheath gas flow 30 Arb, auxiliary gas flow 10 Arb. Metabolite fragments were obtained under high energy collisional dissociation (HCD) mode with a collision energy at 20 eV.

An EASY-nLC 1000 LC system (Thermo Fisher Scientific) interfaced via nanoSpray Flex ion source to an Orbitrap Fusion Lumos MS (Thermo Fisher Scientific) was used for MS and MS/MS analyses. A single analytical column setup using PicoFrit Emitters (New Objectives, 75 µm inner diameter) custom packed with Reprosil-Pure-AQ C18 phase (Dr. Maisch, 1.9-µm particle size, 19–21 cm column length) was applied in nLC. 2 μL of each sample was injected onto the column, followed by elution with a gradient of Solvent B from 2% to 25% at 200 nL/min for 45 min (Solvent A: 100% H₂O+ 0.1% (v/v) formic acid; Solvent B: 100% acetonitrile +0.1% (v/v) formic acid). With the nominal resolution setting of 120,000, precursors of MS1 scan (m/z 350-2,000) were obtained. Then HCD-MS2 of the five most abundant multiply charged precursors in the MS1 spectrum was acquired at the nominal resolution setting of 60,000. To trigger data-dependent fragmentation events, the minimum MS1 signal threshold was 50,000. Targeted MS/MS analysis was performed by setting up a targeted MSn (tMSn) Scan Properties panel. 30 targeted entries were included in the Mass List Table.

Two
microliters per sample (10% of total) was injected per analysis. The
glycans were separated by nanoflow liquid chromatography (nanoLC)
using a single analytical column setup packed with Reprosil-Pure-AQ
C18 phase (Dr. Maisch, 1.9 μm in particle size, 19–21
cm in column length) in an EASY-nLC 1200 UHPLC (Thermo Fisher Scientific)
using a PicoFrit Emitter (New Objectives, 75 μm in inner diameter).
The emitter was interfaced to an Orbitrap Fusion Lumos MS (Thermo
Fisher Scientific) via a nanoSpray Flex ion source. Details on the
LC–MS/MS methods can be found in the Supporting Experimental Section.

Approximately 2 μg samples were analyzed by orbitrap Fusion Lumos MS (Thermo Fisher Scientific, Waltham, MA, USA) coupled online to an EASY-nLC 1200 system with a data-independent acquisition mode (DIA) (24 (link)). The mobile phase used for the liquid chromatography consisted of buffer A (0.1% FA) and buffer B (80% acetonitrile, 0.1% FA). The peptides were separated using a 160-min non-linear gradient consisting of 4–35% buffer B for 85 min, 35–100% buffer B for 60 min and 100% buffer B for 15 min at a flow rate of 300 nl min⁻¹. The acquired DIA data were analyzed by DIA-NN software against the protein database of P. aeruginosa PA14 (25 (link)) as well as E. coli BL21 (DE3). Cysteine carbamidomethylation was searched as the fixed modification, methionine oxidation was collected as the variable modification. Data processing was conducted using the software Perseus. Student's t-test was used to assess the significance of differential expression of proteins (DEPs) between two groups. Proteins that have significance level of P < 0.05 and fold change >1.5 or <–1.5 were considered as DEPs (detailed data in Supplementary Tables S4–S7).

Each sample was analyzed on a Thermo Scientific Orbitrap Fusion Lumos MS via 3 technical replicate injections using a data-dependent acquisition (DDA) HCD MS2 instrument, using the parameters outlined in Supplementary Table S4. The technical replicates were blocked, and each block was randomized. Pooled QCs, which is a mixture of all the samples being analyzed, were analyzed at the start, end, and in between each sample. MS data were analyzed using Proteome Discoverer 2.4 (Thermo) platform as outlined in the Supplementary Table S5. Protein identifications were filtered to include only those proteins identified by two or more unique peptides identified. Mass spectrometry proteomics data have been deposited to the Proteome Xchange Consortium via the PRIDE partner repository with the dataset identifier PXD043245 and 10.6019/PXD043245.